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WO2018125019A2 - Use of some mirnas for the diagnosis and treatment of diseases associated with insulin - Google Patents

Use of some mirnas for the diagnosis and treatment of diseases associated with insulin Download PDF

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WO2018125019A2
WO2018125019A2 PCT/TR2017/050737 TR2017050737W WO2018125019A2 WO 2018125019 A2 WO2018125019 A2 WO 2018125019A2 TR 2017050737 W TR2017050737 W TR 2017050737W WO 2018125019 A2 WO2018125019 A2 WO 2018125019A2
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mir
insulin
cells
disease
group
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WO2018125019A3 (en
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Selda OKTAYOĞLU
Ediz COŞKUN
Merve ERCIN
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Istanbul Ueniversitesi
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Istanbul Ueniversitesi
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/65—MicroRNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to use of certain microRNAs in the diagnosis and treatment of insulin-related diseases such as diabetes and obesity.
  • the invention further relates to use of said microRNAs in the cellular treatment of the insulin-related diseases such as diabetes and obesity, by allowing pancreatic ⁇ -cells to be formed via delivery of microRNAs to stem cells in vitro, and transplanting these cells to the patients.
  • pancreas is a mixed organ consisting of exocrine and endocrine parts.
  • the exocrine part secretes digestive enzymes
  • the endocrine part known as Langerhans islets secretes different hormones.
  • Each islet consists of a large number of specialized endocrine cells and these cells separate from each other due to their specific hormones.
  • An insulin-secreting ⁇ -cell, glucagon- secreting a-cell, somatostatin-secreting ⁇ -cell, and pancreatic polypeptide-secreting PP cells are the four major types of specialized cell types that constitute the islet.
  • the present invention includes at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA molecule thereof, for use in treatment of the insulin-related diseases.
  • Said insulin-related disease may be selected from the group consisting of prediabetes, Type I diabetes mellitus, Type II diabetes mellitus, metabolic syndrome, obesity, lactose intolerance, fructose intolerance, galactosemia, glycogen storage disease, insulin resistance syndrome, syndrome X, retinopathy, neuropathy, nephropathy, foot ulcers, hypertension, hyperlipidemia, metabolic syndrome, gall bladder diseases, osteoarthritis, cardiovascular diseases, stroke, sleep apnea, liver disease, asthma, respiratory distress, menstruation disorders, musculoskeletal system disorders, skin diseases, polycystic ovarian syndrome, immune system disorders, Alzheimer's disease and the other types of dementia, Parkinson's disease and other Parkinson's- related diseases, Huntington's disease, amyotrophic lateral sclerosis (ALS], Batten disease, motor neuron disease, spinal muscular atrophy, Prion disease, spinocerebellar ataxia, lewy body dementia, multiple sclerosis (MS], neuropathy and Friedrich ataxi
  • Molecules of the invention may be in combination with at least one additional active substance selected from antidiabetic, anti-obesity and anti-inflammatory agents.
  • Said antidiabetic agent may be selected from the group consisting of insulin analogs, insulin sensitizers, insulin secretagogues, aldose reductase inhibitors, alpha glucosidase inhibitors, amylin analogs, peptide analogs, sodium glucose transporter 2 (SGLT] inhibitors and glucosuric agents.
  • Said anti-obesity agent may be selected from the group consisting of 4-methyl amphetamine, amfecloral, amfepentorex, amfepramon, aminorex, amphetamine, atomoxetine, benfluorex, benzphetamine, bupropion, cathine, cathinone, chlorphentermine, cyclazindol, clobenzorex, cloforex, clominorex, clotermine, dexfenfluramine, dextroamphetamine, dexmethylphenidate, difemetorex, dimethylcathinone, difemethoxydine, ephedrine, ephedra, ethylamphetamine, etolorex, fenbutrazate, fencamfamin, fenethylline, fenfluramine, fenproporex, fludorex, fluminorex, furfenorex, indan
  • Said anti-inflammatory agent may be selected from the group consisting of pyrazolone/pyrazolidines, salicylates, acetic acid derivatives, oxicams, propionic acid derivatives, N-arylanthranilic acids and coxibs.
  • Molecule of the invention may be presented in a pharmaceutical formulation.
  • the formulation may be in a dosage form that may be administered to a patient orally, rectally, vaginally, intratumorally, subcutaneously, intracutaneously, intravenously, intracerebroventricularly, intramuscularly, intra-arterially, intrathecally, intranasally, interperitoneally, parenterally, topically, or by means of medical devices.
  • the formulation is suitable to be targeted to, or to be carried by, the cells via nanoparticles, liposomes or other carriers.
  • the invention in another aspect, relates to a method for obtaining pancreatic ⁇ -cells in vitro, said method comprising treatment of mesenchymal stem cells with at least one microRNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR- 503, or a DNA, mRNA or a related IncRNA thereof.
  • pancreatic mesenchymal stem cells may be treated with HDAC (Histone deacetylase] inhibitor and/or glucose.
  • Said HDAC inhibitor is preferably valproic acid or a pharmaceutically acceptable salt thereof.
  • HDAC inhibitor has a concentration ranging between 0.5 mM to 5 mM.
  • glucose may have a concentration ranging between 5 mM to 50 mM.
  • the invention relates to pancreatic ⁇ -cells obtained from the method above for use in the treatment of the insulin-related diseases.
  • Said pancreatic ⁇ -cells may be presented in combination with at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or one or more related IncRNA.
  • Said ⁇ -cells may also be provided in combination with at least one additional agent selected from antidiabetic, anti-obesity and anti-inflammatory agents.
  • the invention includes the case in which said ⁇ -cells are provided in a pharmaceutical composition comprising at least one excipient
  • Said pharmaceutical composition is suitable to be targeted to, or to be carried by, the cells via the carriers selected from heparin, lactic acid based polymers, polyesters, hydrogels, biopolymer films, extravascular compartments, intravascular compartments, alginate, poly(hydroxyethylmethacrylate-methyl methacrylate], agarose, acrylonitrile copolimers, chitosan, and PEG nanoparticles and liposomes.
  • the invention in another aspect, relates to a method for in vitro diagnosis of the insulin-related diseases, said method comprising use of at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or one or more related IncRNA as a biomarker.
  • Diseases diagnosed herein are as listed above.
  • the diagnosis method comprises detecting at least one of said biomarkers in a sample selected from the group consisting of blood, plasma, serum, milk, bronchoalveolar fluid and cerebrospinal fluid.
  • the invention relates to a kit for in vitro diagnosis of the insulin-related diseases, preferably the above-mentioned diseases, the kit comprising a biomarker which comprises at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA.
  • the invention provides a kit for in vitro amplification of pancreatic ⁇ -cells, including at least one microRNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof.
  • a kit for in vitro amplification of pancreatic ⁇ -cells including at least one microRNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof.
  • Figure 1 shows the effect of VP A of 0.75-3 mM on PI-MSC viability as % Control.
  • Figure 2 shows the effect of 25mM of Glucose, ImM of VP A, and 25 mM of Glucose+ 1 mM of VP A on the ⁇ -cell differentiation at the end of days 10, 20 and 30. Histogram plots showing flow cytometric insulin + cell number (%, Fluorescence intensity] (A], a graphic showing the analysis results (B], and mean ⁇ SEM values are provided. *p ⁇ 0,05, **p ⁇ 0,01, ***p ⁇ 0,001 vs. control.
  • Figure 3 shows the effect of experimental conditions applied to PI-MSCs for 20 days on mRNA production. A graphic representing the analysis results of mRNA levels (fold increase] (A] and mean ⁇ SEM values (B] are provided. *p ⁇ 0,05; **p ⁇ 0,01 and ***p ⁇ 0,001 vs. control.
  • Figure 4 shows microscopic photographs of the control group received complete medium (a] and the group received glucose (b] at the end of 20-day experiment period.
  • the upper panel is magnified 4X
  • the middle panel is magnified 10X
  • the lower panel is magnified 2 OX. *shows the stem cell colony.
  • Figure 5 shows microscopic photographs of the group received VPA (a] and the group received VPA+glucose (b] at the end of 20-day experiment period.
  • the upper panel is magnified 4X
  • the middle panel is magnified 10X
  • the lower panel is magnified 2 OX. *shows the stem cell colony and ⁇ shows a ⁇ -cell like round cell.
  • Figure 6 shows the effect of experimental conditions applied to PI-MSCs for 20 days on ROS production.
  • Dot plots showing flow cytometric DCF Fluorescence intensity (Fold Increase] (A], a graphic representing the analysis results (B] and mean ⁇ SEM values are provided. **p ⁇ 0,01 and ***p ⁇ 0,001 vs. control, ##p ⁇ 0,01 and ###p ⁇ 0,001 vs. group received glucose.
  • Figure 7 shows insulin amounts (ng/ ⁇ g protein] released from the cells as a result of 5.5 mM of glucose and 25 mM glucose administration. **p ⁇ 0,01 and ***p ⁇ 0,001 vs. control group.
  • Figure 8 demonstrates graphics showing OCT3/4, c-Myc and Nanog protein bands and their intensities in cytoplasmic (left panel] and nuclear (right panel] fractions of PI-MSCs. *p ⁇ 0,05, **p ⁇ 0,01, ***p ⁇ 0,001 vs. control, ##p ⁇ 0,01; ###p ⁇ 0,001 vs. group received glucose, and +p ⁇ 0,05 vs. group received valproic acid.
  • Figure 9 demonstrates graphics showing HDAC1 and HDAC3 protein bands and their intensities in cytoplasmic (left panel] and nuclear (rightpanel] fractions of PI-MSCs. **p ⁇ 0,01, ***p ⁇ 0,001 vs. control, ##p ⁇ 0,01; ###p ⁇ 0,001 vs. group received glucose.
  • Figure 10 demonstrates graphics showing H4-K12 Ace, H3-K9 Ace and H3-K9 TriMe protein bands of PI-MSCs, and band intensities thereof.
  • Figure 11 shows the changes in the expression levels of some miRNAs in the control group and the glucose-treated group. A statistically significant increase was found only in miRNAs that were above the blue line, while red circles show miRNAs, the gene expression levels of which are increased (p ⁇ 0,05].
  • Figure 12 shows the changes in the expression levels of some miRNAs in the control group and the VPA-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased. A statistically significant increase was found only in miRNAs that were above the blue line (p ⁇ 0,05).
  • Figure 13 shows the changes in the expression levels of some miRNAs in the control group and the VPA+glucose-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased. A statistically significant increase was found only in miRNAs that were above the blue line (p ⁇ 0,05).
  • Figure 14 shows the changes in the expression levels of some miRNAs in the glucose-treated group and the VPA+glucose-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased. A statistically significant increase or decrease was found only in miRNAs that were above the blue line (p ⁇ 0,05].
  • Figure 15 shows the changes in the expression levels of some miRNAs in the VPA+glucose-treated group vs. VPA-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased.
  • Figure 16 shows the grade for gene expression level of miRNAs in each group. Gene expression levels increase from green to red.
  • the invention relates to at least one RNA selected from the group consisting of miR- 18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, for use in the treatment of the insulin-related diseases, preferably diabetes and obesity.
  • the present invention relates to use of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof in the treatment of the insulin-related diseases, preferably diabetes and obesity as a medicament in combination with antidiabetic/anti-obesity drugs.
  • the invention relates to use of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, alone or in combination with other antidiabetic, anti-obesity and anti-inflammatory drugs, in order to produce new ⁇ -cells in the cellular treatment of the insulin-related diseases, preferably diabetes and obesity. It is also suggested that more efficient cellular treatment methods can be developed by adding said molecules to the current ⁇ -cell differentiation protocols.
  • the present invention relates to use of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, alone or in combination, or together with known biomarkers in the diagnosis of the insulin-related diseases, preferably diabetes and obesity.
  • mRNA used in the present invention refers to messenger RNAs. The mRNA molecules transfer the genetic information obtained from DNA to the ribosome.
  • the phrases "mRNA” and “messenger RNA” used in the present invention have the same meaning and are used interchangeably.
  • RNA used in the present invention refers to a small and non-coding RNA molecule. These molecules may be used for RNA silencing and regulation of post-transcriptional gene expression.
  • micro RNA and “miRNA” have the same meaning and may be used interchangeably.
  • IncRNA used in the present invention refers to long non-coding RNAs. These RNAs are transcripts that do not code for protein and have a length of more than 200 nucleotides.
  • lnc RNA and “long non-coding RNA” used in the present invention have the same meaning and may be used interchangeably.
  • diabetes mellitus used in the present invention refers to type I diabetes mellitus and/or type II diabetes mellitus.
  • insulin-related diseases used in the present invention refers to various neurodegenerative diseases such as lactose intolerance, fructose intolerance, galactosemia, glycogen storage disease, insulin resistance syndrome, syndrome X, retinopathy, nephropathy, foot ulcers, hypertension, hyperlipidemia, metabolic syndrome, gall bladder diseases, osteoarthritis, cardiovascular diseases, stroke, sleep apnea, liver disease, asthma, respiratory distress, menstruation disorders, musculoskeletal system disorders, skin diseases, polycystic ovarian syndrome, immune system disorders, diabetes mellitus, obesity and Alzheimer's disease and the other types of dementia, Parkinson's disease and other Parkinson's-related diseases, Huntington's disease, amyotrophic lateral sclerosis (ALS], Batten disease, motor neuron disease, spinal muscular atrophy, Prion disease, spinocer
  • ALS amyotrophic
  • MiR-184 is important for the regulation of ⁇ -cell mass increase i.e. compensation, in the case of insulin resistance.
  • Ago2 which is the target of miR-184 in pancreatic islets, is part of the RNA- induced silencing complex required for targeting mRNAs. Loss of Ago2 blocks the compensatory increase of ⁇ -cells, which develops in response to insulin resistance by increasing the expression of miR-375 targets.
  • miR-184 was suppressed in the islets of insulin resistant mice and humans, resulting in increased expression of Ago2, thereby compensatory increase of ⁇ -cells being suppressed.
  • Ago2 levels are decreased and compensatory increase of ⁇ -cells is suppressed.
  • MiR-335 is associated with ⁇ -cell function, rather than ⁇ -cell differentiation.
  • islets were isolated from Goto Kakizaki (GK] rats which were used as a type 2 diabetes mellitus, a ⁇ -cell dysfunction. It has been determined that miR-335 expression is increased in these islets and that their targets are mRNAs of Stxbpl, Sytll and Snap25 which are molecules associated with insulin exocytosis (Esguerra et al., 2011].
  • overexpression of miR-335 leads to a decrease in glucose-induced insulin secretion and a decrease in depolarization-induced insulin exocytosis.
  • VPA-treated groups in which we detected ⁇ -cell differentiation, unresponsiveness to glucose- induced insulin secretion is parallel to the increase in miR-335 expression.
  • MiR-30d is associated with pancreas development, whereas miR-19b is associated with HDCAs.
  • MiR-30 family are produced at high rates in the human fetal pancreas and are associated with epithelial-mesenchymal transition. These miRNAs inhibit mesenchymal mRNAs such as vimentin and Snaill and allow pancreatic mesenchymal stem cells to transform into insulin producing cells. It has been shown that miR-30d, a member of this family, regulates the ⁇ - cell transcription factor MafA, butdoes not affect Pdx-1 and NeuroDl. MiR-30d increased the MafA level and the transcription of the insulin gene.
  • MiR-19b has been reported to show an increase in acute myeloid leukemia cells treated with Vorinostat, a class I and II HDAC inhibitor. However, there was no study showing the relationship of miR-19b with HDAC except this study. It has also been shown that miR-19b is expressed at high levels in pancreatic progenitor cells and that this miRNA targets the 3 'UTR region of NeuroDl mRNA, thereby reducing the protein and mRNA levels of this transcription factor. It has also been shown that MiR- 19b inhibits insl expression in MIN6 cells, does not affect ins2, and has little effect on the proliferation of pancreatic progenitor cells.
  • MiR-124 is one of the best characterized and most abundantly expressed neuronal miRNAs. Overexpression of MiR-124 results in an increase in the expression of neuronal markers, and thus, neurite growth, indicative of neuronal differentiation, has also been shown to manifest itself as morphological changes. In some studies conducted in vertebrates, miR-124 has been identified as a stimulant for neuronal differentiation and as an inhibitor of self-renewal occurred in progenitor cells. However, it is not known how miR-124 directs neurogenesis in mesenchymal stem cells. In the study of the present invention, findings which suggest that miR-124 mediates differentiation of glucose-treated PI-MSCs into ⁇ -cells, were found for the first time in the literature.
  • the invention in another aspect, relates to a method for the formation of pancreatic ⁇ -cell suitable for the treatment of insulin-related diseases, preferably diabetes and obesity, comprising in vitro administration of at least one RNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, to the stem cells.
  • RNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, to the stem cells.
  • the invention relates to use of pancreatic ⁇ -cells obtained by co-administration of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof to the stem cells with at least one other antidiabetic, anti-obesity or anti-inflammatory agent, for the treatment of the insulin-related diseases, preferably for the treatment of diabetes and obesity.
  • at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof to the stem cells with at least one other antidiabetic, anti-obesity or anti-inflammatory agent, for the treatment of the insulin-related diseases, preferably for the treatment
  • the invention relates to use of pancreatic ⁇ -cells obtained by in vitro administration of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR- 30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, to the stem cells in combination with at least one other antidiabetic, anti-obesity or anti-inflammatory agent in the treatment of the insulin-related diseases, preferably in the treatment of diabetes and obesity.
  • RNA selected from the group consisting of miR-18a, miR-19b, miR- 30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, to the stem cells in combination with at least one other antidiabetic, anti-obesity or anti-inflammatory agent in the treatment of the insulin-related diseases,
  • the present invention also relates to a method for use in the diagnosis of the insulin-related diseases, preferably diabetes and obesity, comprising detecting the presence of at least one of biomarker comprising at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, in all body fluids such as blood, plasma, serum, milk, bronchoalveolar fluid and cerebrospinal fluid.
  • biomarker comprising at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, in all body fluids such as blood, plasma, serum, milk, bronchoalveolar fluid and cerebrospinal fluid
  • Said molecules may be determined by precise molecular methods such as Southern Blot, Northern Blot, PCR, RT-PCR, qRT-PCR, microarray and sequencing and the methods that may be used are not limited to said techniques.
  • Antidiabetic agent used in the present invention means agents used in the treatment of diabetes mellitus, whereas "anti-obesity agent” refers to agents used in the treatment of obesity. All molecules (alone or in combination], all the related RNAs and DNAs, which were described in the present invention and detailed above can be used in cellular therapy in conjunction with antidiabetic, anti-obesity or anti-inflammatory drugs.
  • Said antidiabetic agents may be selected from insulin analogs, insulin sensitizers, insulin secretagogues, aldose reductase inhibitors, alpha glucosidase inhibitors, amylin analogs, peptide analogs, sodium glucose transporter 2 (SGLT] inhibitors and glucosuric agents.
  • Insulin that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of insulin, insulin lispro, insulin aspart, insulin glulisine, insulin zinc, isophane insulin, insulin glargine, insulin detemir.
  • Insulin sensitizers that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of metformin, phenformin, buformin, siglitazone, darlitazone, englitazone, lobeglitazone, netoglitazone, rivoglitazone, aleglitazar, saroglitazar, tesaglitazar, rosiglitazone, pioglitazone, and troglitazone.
  • Insulin secretagogues that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of acetohexamide, carbutamide, chlorpropamide, metahexamide, tolbutamide, tolazamide, glibenclamide, glibornuride, glicetanyl, gliclazide, gliflumide, glipizide, gliquidone, glisoxepide, glyclopyramide, glimepiride, repaglinide, mitiglinide, exenatide, liraglutide, taspoglutide, albiglutide, lixisenatide, dulagutide, semaglutide, alogliptin, anagliptin, gemigliptin, linagliptin, omarigliptin, saxagliptin, sitagliptin, tenegliptin, vildagliptin, fasiglif
  • Aldose reductase inhibitors that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of epalrestat, fidarestat, ranirestat, tolrestat, and zenarestat.
  • Alpha glucosidase inhibitors that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of miglitol, acarbose, and voglibose.
  • Amylin analog that can be used in combination with the molecules listed above in the context of the present invention is pramlintide.
  • Sodium glucose transporter 2 (SGLT] inhibitors that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of canaglifozin, dapagliflozin, empagliflozin, remogliflozin, sergliflozin, and tofogliflozin.
  • the antidiabetic agent may also be benfluorex or bromocriptine.
  • Said anti-obesity agents may be selected from the group consisting of 4-methyl amphetamine, amfecloral, amfepentorex, amfepramon, aminorex, amphetamine, atomoxetine, benfluorex, benzphetamine, bupropion, cathine, cathinone, chlorphentermine, cyclazindol, clobenzorex, cloforex, clominorex, clotermine, dexfenfluramine, dextroamphetamine, dexmethylphenidate, difemetorex, dimethylcathinone, difemethoxydine, ephedrine, ephedra, ethylamphetamine, etolorex, fenbutrazate, fencamfamin, fenethylline, fenfluramine, fenproporex, fludorex, fluminorex, furfenorex, indan
  • Said anti-inflammatory agents may be selected from the general groups consisting of pyrazolone/pyrazolidines, salicylates, acetic acid derivatives, oxicams, propionic acid derivatives, N-arylanthranilic acids, coxibs and the other agents.
  • Said anti-inflammatory agents may be selected from the groups consisting of aminophenazone, ampyrone, clophenazone, famprofazone, feprazone, kebuzone, metamizole, mofebutazone, morazone, nifenazone, oxyphenbutazone, phenazone, phenylbutazone, propyphenazone, sulfinpyrazone, suxibuzone, acetylsalicylic acid, aloxypyrine, benorilate, carbasalate calcium, diflunisal, ethenzamide, guacetical, magnesium salicylate, methyl salicylate, salsalate, salicylamide, salicylic acid, sodium salicylate, aceclofenac, acemetacin, alclofenac, amfenac, bendazac, bromfenac, bumadizone, bufexamac, diclofenac, difenpiramide, etodolac
  • the invention relates to pharmaceutical compositions comprising ⁇ -cells obtained by the methods according to the invention.
  • Said pharmaceutical compositions may contain at least one other excipient in addition to the beta cells.
  • the pharmaceutical compositions according to the invention may contain at least one other active ingredient in addition to the ⁇ - cells.
  • the other active ingredient may be selected from the antidiabetic or anti-obesity agents described in detail above, or a combination thereof.
  • the other active agent may be formulated together with ⁇ -cells, or it may be formulated separately and administered to the patient simultaneously, sequentially or at different times.
  • compositions according to the present invention are administered to the culture medium in vitro to form beta cells, these cells can be injected intravenously.
  • Formulations suitable for injection may be formulated using a sterile solvent or any pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be selected from sterile water, saline, or the existing cell culture media within the scope of state of the art, but is not limited thereto.
  • compositions according to the present invention may be administered parenterally in the form of an injectable formulation.
  • Formulations suitable for injection may be formulated using a sterile solvent or any pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be selected from sterile water, saline, or the existing cell culture media within the scope of state of the art, but is not limited thereto.
  • the invention relates to a method of ⁇ -cell differentiation, comprising a step of obtaining ⁇ -cells by administrating a HDAC (Histone deacetylase] inhibitor and glucose to pancreatic mesenchymal stem cells.
  • HDAC Histone deacetylase
  • valproic acid or a pharmaceutically acceptable salt thereof, such as sodium valproate is used as the HDAC inhibitor.
  • the HDAC inhibitor is used in a range of 0.5 mM to 5 mM, preferably in a range of 0.75 mM to 3 mM, particularly preferably in a range of 1 mM to 2 mM.
  • the ⁇ -cell differentiation protocol uses glucose in a range of 5 mM to 50 mM, preferably in a range of 10 nM to 40 mM, particularly preferably in a range of 20 mM to 30 mM.
  • the invention relates to pharmaceutical formulations comprising at least one RNA selected from the group of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR- 335, miR-433-5p ve miR-503, or a DNA, mRNA or IncRNA thereof.
  • compositions according to the invention may, in addition to said RNAs, contain at least one pharmaceutically acceptable excipient and/or additional active ingredient
  • the other active agent mentioned herein may be an antidiabetic agent or an anti-obesity agent or anti-inflammatory agent Details of these agents are given in the context of the invention.
  • the formulations according to the invention may be prepared in the form of any of the existing dosage forms known in the art of composition. Said dosage form may be administered to a patient orally, rectally, vaginally, intratumorally, subcutaneously, intracutaneously, intravenously, intracerebroventricularly, intramuscularly, intraarterially, intratracheally, interperitoneally, parenterally, topically, or by means of medical devices.
  • the formulations according to the present invention can be suitably formulated to be targeted to, or to be carried by, the cells via nanoparticles, liposomes and the other similar carriers, or they can be administered by said routes.
  • the formulations according to the present invention may be formulated in a manner suitable for administration to the patient by nasal, spray, oral, aerosol, rectal or vaginal route of administration, or they can be administered to the patient by said routes.
  • compositions according to the present invention may be administered parenterally in the form of an injectable formulation.
  • Formulations suitable for injection may be formulated using a sterile solvent or any pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be selected from sterile water, saline, or the existing cell culture media within the scope of state of the art, but is not limited thereto.
  • Example 1 Materials and Methods Used in the Experimental Procedures
  • pancreatic islet-derived mesenchymal stem cells used in this study were isolated from adult (2.5-3 months of age] Wistar albino rats and characterization studies were completed. All cell culture studies were carried out in Istanbul University, Faculty of Sciences, Department of Biology, Department of Molecular Biology, Primer and Stem Cell Research Laboratory. PI-MSCs between 6 and 9 passages were used in this study.
  • MEM Minimum Essential Medium
  • FBS heat-inactivated fetal bovine serum
  • an antibiotic mixture 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, Gibco-15240-122
  • glutamin (2mM, Gibco, 25030] glutamin 2mM, Gibco, 25030]
  • complete medium at 37°C, 5% C02 and 95% air at a humidified incubator, known as standard culture conditions.
  • Cells reproduced in sterile culture dishes were passaged after filling at least 80% of the culture dish surface. During this process, the cells were allowed to remove from the culture dish by applying 0,25% trypsin-EDTA (Sigma-T4174]. After the trypsin was inactivated by adding complete medium, the cells were collected by centrifuging at 1500 rpm for 5 minutes, and a plating process was performed in a new culture dish after cell counting.
  • PI-MSCs Four different experimental conditions were applied to PI-MSCs. These are i] a complete medium which is a normal growing and developing medium, ii] a complete medium containing 25 mM of glucose, iii] a complete medium containing 1 mM of Valproic acid, and iv] a complete medium containing 1 mM of Valproic acid+25 mM of glucose.
  • D-(+]-Glucose Sigma G7021] was preferred to create glucose-containing conditions and sodium valproate (Sanofi Aventis, Depakine] was used for valproic acid-containing conditions.
  • MTT (3-(4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide] test is a toxicity test aimed at directly assessing cell viability and indirectly cell death.
  • the basis of this method is the reduction of the tetrazolium ring of MTT to a blue-violet, water-insoluble formazan by succinate dehydrogenase, a mitochondrial enzyme, in viable cells.
  • the capability of the cells to reduce MTT is accepted as a criterion of cell viability and formazan density obtained as a result of this reaction is directly proportional to the number of viable cell.
  • Viable cells, the mitochondrial function of which is undistorted, are stained in purple color, whereas dead cells or cells having impaired mitochondrial function are not stained.
  • DMSO dimethylsulfoxide
  • PI-MSC PI-MSC
  • PI-MSC PI-MSC
  • VPA 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, selected in accordance with the literature was applied for 24 hours to determine non-toxic doses of VPA. (Haumaitre et al., 2008].
  • Cells grown in a complete medium were used as a control.
  • 50 mg of MTT (Applichem, A2231 0001] were sterilized by passing through a filter of 0.22 ⁇ after bringing to 10 ml with MEM and dissolving.
  • RNA level was measured in a Qubit Fluorimeter (Invitrogen] using the Qubit® RNA HS Assay Kit (Thermofisher, Q32852].
  • RNA was converted to cDNA by reverse transcriptase (NEB, ProtoScript First Strand cDNA Synthesis Kit, E6300S].
  • NEB reverse transcriptase
  • RNA denaturation was performed at 70°C for 5 minutes.
  • incubation was carried out for 1 hour at 42°C followed by incubation at 80°C for 5 minutes in order to stop the enzyme activity.
  • Thermal Cycler device was used for these steps.
  • Total cDNA level was measured in a Qubit Fluorimeter (Invitrogen] using the Qubit ssDNA Assay Kit.
  • the ability of these cells to release insulin in response to increase in glucose was measured.
  • the cells were incubated in DMEM medium containing 5.5 mM/L of glucose and 0.5% BSA for two hours subsequent to washing with PBS. After the culture medium was collected, the cells were washed with PBS and incubated for two hours in DMEM medium containing glucose (25 mM/L) at high concentration. Insulin levels of the collected media were determined by sandwich ELISA technique and the protocol provided by the kit (Millipore, EZRMI- 13K]. The total protein content of the cells was calculated by the Bradford test and a normalization process was performed proportioning the insulin levels to the protein levels.
  • DCFDA 2',7'-dichlorofluorescein diacetate
  • ROS reactive oxygen species
  • ROS level changes between experimental groups were determined by flow cytometry using DCFDA. Analyses were performed with 50,000 cells from each group and repeated at least 3 times. DCFDA (Sigma-D6883] was added to the cells at a concentration of 10 ⁇ , incubated for 30 min in the dark at standard culture conditions and analyzed on FLl channel on a flow cytometer (BD, FACS Calibur]. The mean fluorescence intensity from the control group was taken as 1 and the ROS levels in the other groups were calculated as fold increase relative to the control group.
  • Example 5 Western Blot The lysis buffer (Invitrogen, FNN0011], to which the protease-phosphatase inhibitor mixture (Serva-39055] was added, was introduced to the cell pellet. This was allowed to stand for 30 minutes with stirring once every 10 minutes on ice. Ultrasonication was performed 5 times for 10 seconds. After centrifugation at 13000 rpm at °C for 10 minutes, the supernatant was stored in a freezer at -86°C for use as a cytoplasmic fraction.
  • lysis buffer Invitrogen, FNN0011]
  • the protease-phosphatase inhibitor mixture Serva-39055
  • the lysis buffer (Invitrogen, FNN0011], to which the protease-phosphatase inhibitor mixture (Serva-39055] was added, was introduced to the cell pellet. This was allowed to stand for 30 minutes with stirring once every 10 minutes on ice. Ultrasonication was performed 5 times for 10 seconds. After centrifugation at 13000 rpm at 4°C for 10 minutes, the supernatant was stored in a freezer at -86°C for use as a cytoplasmic fraction.
  • Samples containing 30 ⁇ g protein for the cytoplasmic fraction and 10 ⁇ g protein for the nuclear fraction were prepared in a volume of 10 ⁇ , and denaturation process was carried out by leaving a total of 20 ⁇ of the mixture obtained by adding 10 ⁇ of laemmli buffer, in a dry block heater heated to 100°C (Techne Dri-Block DB-2D] for 5 minutes. After a brief centrifugation, the wells of the prepared gels were loaded. 25 mM Tris, 192 mM Glisin, 0,1% SDS; pH:8,3 were used a the running buffer. Proteins were run at 70 V for 30 minutes, 100 V for 45 minutes, and at 120 V and +4°C until the running process was terminated. D) Transfer of Proteins from Gel to a Membrane
  • Proteins separated by gel electrophoresis were transferred from the gel to a PVDF (Polyvinylidene fluoride] membrane by wet transfer.
  • PVDF Polyvinylidene fluoride
  • Membranes (Millipore-Immobilon-PSQ] with a pore diameter of 0.2 ⁇ were used for the transfer of proteins less than 20 kDa, while membranes with a pore diameter of 0.45 ⁇ (Millipore-Immobilon-P] were preferred for the transfer of proteins larger than 20 kDa. Transfer was carried out in Tris-Glycine SDS buffer containing 20% methanol for 12 hours at a current intensity of 80mA and + 4°C.
  • Ponceau S staining solution was applied to the membrane for 5 min, and the efficiency of the transfer was checked by observing the band densities. After the bands were observed, 0.1 M NaOH was applied for 5 min to remove the Poncaue S stain from the membrane.
  • the membrane was incubated with a blocking solution prepared with 5% skimmed milk powder for 1 hour at room temperature in a shaker. Membranes were incubated overnight at +4°C with primer antibodies (Table 3.2] prepared with appropriate dilution rate after blocking. After this process, the membranes washed with the washing buffer TBS-T (95% TBS, 5% Twin-20] were treated with the secondary antibodies specific for HRP (horseradish peroxidase]-labeled primer antibody (Table 3.2] for 1 hour at room temperature.
  • HRP horseradish peroxidase
  • miRNAs associated with pluripotency, early pancreatic development, end term pancreas maturation, Type 2 diabetes mellitus and HDACs have been determined.
  • U6 was used as an internal control. Results were calculated in 2 ACT values.
  • 700 ul of this mixture was transferred to 2 ml of collection tubes with filters. Centrifugation was performed at 8000xg for 15 seconds. 700 ul of RWT buffer was added to the columns. Centrifugation was performed at 8000xg at room temperature for 15 seconds. 500 ⁇ of RPE buffer was added and centrifuged at 8000xg at room temperature for 15 seconds. Again, 500 ⁇ of RPE buffer was added and centrifuged at 8000xg at room temperature for 2 minutes. The column was placed in eppendorf tubes of 1.5 ml. 50 ⁇ of Rnase-free water was added to the column. The tube was centrifuged at 8000xg for 1 minute after sealing. miRNAs were allowed to be transferred to the tube together with water.
  • the Qubit® RNA HS Assay Kit (Thermofisher, Q32852] was used to quantify the amount of RNAs obtained as a result of isolation. Tubes of 0.5 ml were prepared for the standards and samples. Solution required for the samples and standards being studied by diluting Qubit RNA HS Reagent in Qubit RNA HS Buffer at a ratio of 1 :200. 190 ⁇ and 199 ⁇ of this solution were transferred to the standard tubes and the sample tubes, respectively. RNA levels were determined by Qubit Fluorimeter 2.0 (Invitrogen] after 10 ⁇ of standards and 1 ⁇ of samples were added, followed by incubation at room temperature for 5 min in the dark.
  • RNA was converted to cDNA using reverse transcriptase enzyme (Qiagene, 2180 3]. For each sample, a total of 20 ⁇ of mixture was formed. This mixture was composed of 4 ⁇ of 5x miScript buffer, 2 ⁇ of lOx miScript mix, 2 ⁇ of miScript Reverse Transcriptase, 5.75 ⁇ of RNase-free water, and 6.25 ⁇ of RNA.
  • incubation was carried out at 95°C for 5 minutes to inactivate the reverse transcriptase enzyme after incubation at 37°C for 60 minutes. Thermal Cycler device (Techne] was used for these steps.
  • reaction mixture prepared in a total volume of 20 ⁇ was composed of 4.5 ⁇ of RNase-free water (QIAGEN], 10 ⁇ of SYBR green (2xQuaintiTect SYBR Green PCR], 2 ⁇ of master mix (lOx miScript Universal Primer], 2 ⁇ of primer mix QIAGEN, miscript primer Assays] and 1.5 ⁇ of cDNA.
  • a reaction mixture of 1.5 ⁇ without Rnase-free water was used as a negative control instead of cDNA.
  • U6 miRNA was used as an internal control.
  • qRT-PCR analysis was performed using the Rotor-Gene Q Series Software 2.3.1 (Qiagen] program.
  • Valproic acid was applied to PI-MSCs at a dose of 0.75-3 mM for 24 hours.
  • the effect of valproic acid on cell viability at the specified dose range was determined by MTT technique and the results are shown in Figure 1.
  • Cell viability values are as follows: 100 ⁇ 0.00% in the control group; 131.40 ⁇ 1.53 in the group treated with 0.75 mM of VP A; 107.50 ⁇ 1.76 in the group treated with 1 mM of VP A; 91.58 ⁇ 2.52 in the group treated with 1.5 mM of VP A; 76.48 ⁇ 2.12 in the group treated with 2 mM of VP A; 58.28 ⁇ 2.08 in the group treated with 2.5 mM of VP A; and 51.63 ⁇ 2.17 in the group treated with 3 mM of VPA. There was found a significant increase in the number of the cells only in the group treated with 0.75 mM of VPA versus control group.
  • VPA concentrations increased after the group treated with 1 mM of VPA. From these results, it was decided to continue with ⁇ -cell differentiation studies by applying 0.75 mM and 1 mM of VPA that are doses increasing or decreasing the cell viability.
  • the insulin + cell ratios recorded after administration of complete media, 25 mM Glucose, 1 mM VPA, and 1 mM VPA + 25 mM Glucose to PI-MSCs for 10, 20 and 30 days are shown in Figure 2.
  • a significant ⁇ -cell differentiation was observed for the group receiving only 1 mM VPA.
  • a significant ⁇ -cell differentiation was observed for the groups receiving 25 mM Glucose, 1 mM VPA and 1 mM VPA+25mM Glucose. It was found that the highest ⁇ -cell differentiation occurred in the groups for which the application was performed for 20 days. From these results, it was decided for the study to be continued with a 20-day experiment period.
  • Figures 4 and 5 show photographs taken at the end of 20 days on the inverted light microscope.
  • the morphological similarity between the glucose-treated PI-MSCs and the complete medium is remarkable.
  • the common characteristic of both groups is the formation of stem cell colonies of largely varying sizes ( Figure 4].
  • This image corresponds to the characteristic of pluripotent cell.
  • the presence of round cells concentrated in colony-like structures in the PI-MSCs treated with VPA and VPA + glucose is noteworthy. It has been observed that the PI-MSCs surrounding the colonies of such cells have decreased in number such that the surrounding of the colonies is emptied and generally, the round cells separated from these colonies are spread to these spaces.
  • ROS reactive oxygen species
  • insulin release was as follows: 0.15 ⁇ 0.01 ng ⁇ g protein in the control group; 0.29 ⁇ 0.05 ng ⁇ g protein in VPA-treated group; and 0.28 ⁇ 0.01 ng ⁇ g protein in VPA+glucose-treated group.
  • the groups were compared with each other, a significantly increased level of insulin secretion was observed in the VPA and VPA + glucose- treated groups compared to the control group (p ⁇ 0,01].
  • insulin release was as follows: 0.15 ⁇ 0.01 ng ⁇ g protein in the control group; 0.28 ⁇ 0.01 ng ⁇ g protein in the glucose group; 0.28 ⁇ 0.01 ng ⁇ g protein in VPA-treated group; and 0.30 ⁇ 0.01 ng ⁇ g protein in VPA+glucose-treated group.
  • the groups were compared with each other, a significantly increased level of insulin secretion was observed in all groups compared to the control group (p ⁇ 0,01 for G and VPA; p ⁇ 0,001 for VPA+glucose].
  • Example 9 Results for Changes in Pluripotent Character
  • Amount of 0CT3/4 in the cytoplasm was found to be 1.17 ⁇ 0.06 for the control group; 0.69 ⁇ 0.18 for glucose-treated group; 0.32 ⁇ 0.10 for VPA-treated group; and 0.37 ⁇ 0.09 for VPA+G-treated group.
  • Nuclear 0CT3/4 ratios were found to be 0.91 ⁇ 0.16 for the control group; 0.45 ⁇ 0.078 for glucose-treated group; 0.34 ⁇ 0.07 for VPA-treated group; and 0.37 ⁇ 0.02 or VPA+G-treated group.
  • Amount of cytoplasmic c-Myc were calculated as 1.07 ⁇ 0.04 for the control group; 0.60 ⁇ 0.015 for glucose-treated group; 0.36 ⁇ 0.06 for VPA-treated group; and 0.52 ⁇ 0.06 for VPA+G-treated group. Ratios of nuclear c-Myc were found to be 0.30 ⁇ 0.06 for the control group; 0.48 ⁇ 0.09 for glucose- treated group; 0.11 ⁇ 0.07 for VPA-treated group; and 0.07 ⁇ 0.01 for VPA+G-treated group. When cytoplasmic c-myc levels were compared, a significant decrease was found in all groups compared to the control group (p ⁇ 0,001].
  • the amount of cytoplasmic Nanog was 1.32 ⁇ 0.21 for the control group; 0.74 ⁇ 0.07 for glucose- treated group; 0.1 ⁇ 0.01 for VPA-treated group; and 0.12 ⁇ 0.01 for VPA + G-treated group.
  • Ratios of nuclear Nanog were 1.27 ⁇ 0.24 for the control group; 0.76 ⁇ 0.07 for glucose-treated group; 0.19 ⁇ 0.01 for VPA-treated group; and 0.13 ⁇ 0.01 for VPA + G-treated group.
  • PI- MSCs Complete medium, 1 mM VP A, 25 mM Glucose and 1 mM VPA+25 mM Glucose was applied to PI- MSCs for 20 days, with the media being changed daily. Subsequently, cytoplasmic and nuclear fractions were obtained from cell lysates and levels of HDACl and 3, H3K9-Ace, H3K9-Met, H4K12-Ace were determined by western blot method. Optical density values of the protein bands were normalized by proportioning to the ⁇ -actin density for the cytoplasmic fraction and to the TF-IIB density for the nuclear fraction.
  • Figure 9 shows the changes in cytoplasmic and nuclear fraction levels of HDACl and 3 among Class I HDACs. Ratios of cytoplasmic HDACl were 0.14 ⁇ 0.01 for the control group; 0.12 ⁇ 0.02 for glucose-treated group; 0.04 ⁇ 0.01 for VPA-treated group; and 0.03 ⁇ 0.01 for VPA + G-treated group. When the results were compared, a significant decrease was found in VPA- and VPA + G- treated groups compared to the control group (p ⁇ 0,001]. When VPA- and VPA + G-treated groups were compared to glucose-treated group, a significant decrease was detected (p ⁇ 0,001].
  • Ratios of nuclear HDACl were 0.43 ⁇ 0.12 for the control group; 0.31 ⁇ 0.07 for glucose-treated group; 0.09 ⁇ 0.05 for VPA-treated group; and 0.07 ⁇ 0.02 for VPA + G-treated group.
  • a significant decrease was detected in VPA- and VPA + G-treated groups compared to the control group (p ⁇ 0,001].
  • VPA- and VPA + G-treated groups were compared to glucose- treated group, a significant decrease was found (p ⁇ 0,01].
  • Ratios of cytoplasmic HDAC3 were 0.15 ⁇ 0.03 for the control group0.12 ⁇ 0.02for glucose-treated group; 0.05 ⁇ 0.01 for VPA-treated group; and 0.04 ⁇ 0.00 for VPA + G-treated group.
  • a significant decrease was observed in VPA- and VPA+G-treated groups compared to glucose-treated group (p ⁇ 0,001].
  • VPA-treated group was compared to glucose-treated group, a significant decrease was found (p ⁇ 0,01].
  • VPA+G-treated group was compared to glucose-treated group, a significant decrease was found (p ⁇ 0,001].
  • Ratios of nuclear HDAC3 were 0.14 ⁇ 0.01 for the control group; 0.11 ⁇ 0.07 for glucose-treated group; 0.03 ⁇ 0.00 for VPA-treated group; and 0.03 ⁇ 0.00 for VPA + G-treated group. A significant decrease was observed in VPA- and VPA+G-treated groups compared to the control group (p ⁇ 0,01]. When VPA- and VPA+G-treated groups were compared to glucose-treated group, a significant decrease was observed (p ⁇ 0,01].
  • Figure 10 shows the results of histone modifications. H4K12-Ace ratios were calculated as 0.006 ⁇ 0.001 in the control group; 0.005 ⁇ 0.001 in VPA-treated group; and 0.075 ⁇ 0.019 in VPA+G- treated group.
  • VPA- treated group compared to the control and glucose-treated group (p ⁇ 0,01).
  • VPA+G-treated group was compared to the control and glucose-treated groups, a significant increase was observed (p ⁇ 0,05). No significant difference was found between VPA- treated group and VPA+G- treated group (p>0,05).
  • H3K9-Met levels were calculated as 0.225 ⁇ 0.001 in the control group, 0.160 ⁇ 0.283 in glucose- treated group, 0.070 ⁇ 0.001 in VPA-treated group; and 0.080 ⁇ 0.042 in VPA + G-treated group. A significant decrease was observed only in VPA- and VPA+G-treated groups compared to the control group (p ⁇ 0,05).
  • H3K9-Ace levels were calculated as 0.076 ⁇ 0.004in the control group, 0.108 ⁇ O.OOlin glucose- treated group, 0.171 ⁇ 0.001 in VPA-treated group; and 0.119 ⁇ 0.130 in VPA + G-treated group.
  • a significant increase was observed in VPA-treated group compared to the control and glucose- treated group (p ⁇ 0,001].
  • a significant decrease (p ⁇ 0.05] was found when VPA+G-treated group was compared with VPA-treated group, and a significant increase (p ⁇ 0,05] was found when VPA+G-treated group was compared with the control group.

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Abstract

The present invention relates to the use of at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, in the diagnosis and treatment of insulin-related diseases such as diabetes mellitus and obesity.

Description

USE OF SOME MIRNAs FOR THE DIAGNOSIS AND TREATMENT OF DISEASES ASSOCIATED
WITH INSULIN
Technical Field
The present invention relates to use of certain microRNAs in the diagnosis and treatment of insulin-related diseases such as diabetes and obesity. The invention further relates to use of said microRNAs in the cellular treatment of the insulin-related diseases such as diabetes and obesity, by allowing pancreatic β-cells to be formed via delivery of microRNAs to stem cells in vitro, and transplanting these cells to the patients.
State of the Art
In recent years, a significant increase in the incidence of metabolic diseases has been observed worldwide. A sedentary lifestyle and unhealthy eating habits cause obesity and Type 2 diabetes mellitus (T2DM] to develop at a high rate. According to the World Health Organization (WHO], there are 1.4 billion overweight adults and approximately 500 million adults that are obese worldwide. This demonstrates how important to define obesity-related metabolic diseases accurately to be protected from T2DM. The formation process of the pancreatic cells, which play a key role in the development of obesity and T2DM, and the well-defined signal pathways and epigenetic factors involved in this process will be the basis for the treatment of these diseases. Pancreas is a mixed organ consisting of exocrine and endocrine parts. While the exocrine part secretes digestive enzymes, the endocrine part known as Langerhans islets secretes different hormones. Each islet consists of a large number of specialized endocrine cells and these cells separate from each other due to their specific hormones. An insulin-secreting β-cell, glucagon- secreting a-cell, somatostatin-secreting δ-cell, and pancreatic polypeptide-secreting PP cells are the four major types of specialized cell types that constitute the islet.
The global increase in diabetes mellitus requires the development of new therapeutic strategies such as increasing the number of pancreatic β-cells or regenerative biology. Research has been focusing on cellular therapy in recent years, as the exogenous insulin or hypoglycemic agent applications among the therapies for both types of diabetes are not therapeutic and are insufficient to prevent the development of secondary complications associated with diabetes. The proposed treatment for cellular therapy is limited by the small number of β-cells obtained from the pancreas of cadavers and the development of post-transplant immunoreaction. In recent years, studies have focused on re-transplantation of mesenchymal stem cells (MSC] isolated from a patient into the patient by differentiating them into β-cells. As an alternative approach to cell transplantation, it can be considered that endogenous β-cell mass in the pancreas of the patient is increased by applying growth factors. The mechanism that regulates pancreatic β-cell mass should be clarified in order to apply both approaches.
Given the state of the art and the new approaches adopted in treatments, it appears that there is a need to develop methods and agents to provide beta cells in vitro for the treatment of diabetes mellitus and the related metabolic diseases. The treatment makes it necessary to use different molecules, because the targeted diseases are very diverse and the disease inherently has complex mechanisms.
Summary of the Invention In one aspect, the present invention includes at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA molecule thereof, for use in treatment of the insulin-related diseases.
Said insulin-related disease may be selected from the group consisting of prediabetes, Type I diabetes mellitus, Type II diabetes mellitus, metabolic syndrome, obesity, lactose intolerance, fructose intolerance, galactosemia, glycogen storage disease, insulin resistance syndrome, syndrome X, retinopathy, neuropathy, nephropathy, foot ulcers, hypertension, hyperlipidemia, metabolic syndrome, gall bladder diseases, osteoarthritis, cardiovascular diseases, stroke, sleep apnea, liver disease, asthma, respiratory distress, menstruation disorders, musculoskeletal system disorders, skin diseases, polycystic ovarian syndrome, immune system disorders, Alzheimer's disease and the other types of dementia, Parkinson's disease and other Parkinson's- related diseases, Huntington's disease, amyotrophic lateral sclerosis (ALS], Batten disease, motor neuron disease, spinal muscular atrophy, Prion disease, spinocerebellar ataxia, lewy body dementia, multiple sclerosis (MS], neuropathy and Friedrich ataxia. The disease is preferably selected from prediabetes, diabetes mellitus, metabolic syndrome and obesity. More preferably, the disease is Type I diabetes mellitus or Type II diabetes mellitus. The disease may also be prediabetes, an early stage of diabetes.
Molecules of the invention may be in combination with at least one additional active substance selected from antidiabetic, anti-obesity and anti-inflammatory agents. Said antidiabetic agent may be selected from the group consisting of insulin analogs, insulin sensitizers, insulin secretagogues, aldose reductase inhibitors, alpha glucosidase inhibitors, amylin analogs, peptide analogs, sodium glucose transporter 2 (SGLT] inhibitors and glucosuric agents. Said anti-obesity agent may be selected from the group consisting of 4-methyl amphetamine, amfecloral, amfepentorex, amfepramon, aminorex, amphetamine, atomoxetine, benfluorex, benzphetamine, bupropion, cathine, cathinone, chlorphentermine, cyclazindol, clobenzorex, cloforex, clominorex, clotermine, dexfenfluramine, dextroamphetamine, dexmethylphenidate, difemetorex, dimethylcathinone, difemethoxydine, ephedrine, ephedra, ethylamphetamine, etolorex, fenbutrazate, fencamfamin, fenethylline, fenfluramine, fenproporex, fludorex, fluminorex, furfenorex, indanorex, khat, levopropylhexedrine, lisdexamfetamine, manifaxine, mazindol, mefenorex, methamphetamine, methylphenidate, norfenfluramine, pemoline, pentorex, phendimetrazine, phenethylamine, phenmetrazine, phentermine, phenylpropanolamine, psiloreks, pipradrol, prolintane, propylhexedrine, pseudoephedrine, pyrovalerone, radafaxine, reboxetine, setazindol, sibutramine, synephrine, tesofensine, viloxazine, xylopropamine, zylofuramine, drinabant, ibipinabant, otenabant, rimonabant, rosonabant, surinabant, taranabant, 5-HTP, galactomannan, glucomannan, L-DOPA, L-phenylalanine, L-tryptophan, L-tyrosine, lorcaserin, Lu AA-33810, naltrexone, naloxone, oxyntomodulin, P57, peptide YY, topiramate, yohimbine, zonisamide, cetilistat, 2,4-dinitrophenol, dirlotapide, miltratapide, oleoyl estron, orlistat, simmondsin and sterculia. Said anti-inflammatory agent may be selected from the group consisting of pyrazolone/pyrazolidines, salicylates, acetic acid derivatives, oxicams, propionic acid derivatives, N-arylanthranilic acids and coxibs.
Molecule of the invention may be presented in a pharmaceutical formulation. The formulation may be in a dosage form that may be administered to a patient orally, rectally, vaginally, intratumorally, subcutaneously, intracutaneously, intravenously, intracerebroventricularly, intramuscularly, intra-arterially, intrathecally, intranasally, interperitoneally, parenterally, topically, or by means of medical devices. The formulation is suitable to be targeted to, or to be carried by, the cells via nanoparticles, liposomes or other carriers.
In another aspect, the invention relates to a method for obtaining pancreatic β-cells in vitro, said method comprising treatment of mesenchymal stem cells with at least one microRNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR- 503, or a DNA, mRNA or a related IncRNA thereof.
In the method herein, pancreatic mesenchymal stem cells may be treated with HDAC (Histone deacetylase] inhibitor and/or glucose. Said HDAC inhibitor is preferably valproic acid or a pharmaceutically acceptable salt thereof. HDAC inhibitor has a concentration ranging between 0.5 mM to 5 mM. Similarly, glucose may have a concentration ranging between 5 mM to 50 mM.
In another aspect, the invention relates to pancreatic β-cells obtained from the method above for use in the treatment of the insulin-related diseases. Said pancreatic β-cells, optionally and usually for the protection of these cells, may be presented in combination with at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or one or more related IncRNA. Said β-cells may also be provided in combination with at least one additional agent selected from antidiabetic, anti-obesity and anti-inflammatory agents.
In another aspect, the invention includes the case in which said β-cells are provided in a pharmaceutical composition comprising at least one excipient Said pharmaceutical composition is suitable to be targeted to, or to be carried by, the cells via the carriers selected from heparin, lactic acid based polymers, polyesters, hydrogels, biopolymer films, extravascular compartments, intravascular compartments, alginate, poly(hydroxyethylmethacrylate-methyl methacrylate], agarose, acrylonitrile copolimers, chitosan, and PEG nanoparticles and liposomes. In another aspect, the invention relates to a method for in vitro diagnosis of the insulin-related diseases, said method comprising use of at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or one or more related IncRNA as a biomarker. Diseases diagnosed herein are as listed above. The diagnosis method comprises detecting at least one of said biomarkers in a sample selected from the group consisting of blood, plasma, serum, milk, bronchoalveolar fluid and cerebrospinal fluid. In this method, said biomarkers may be detected by a molecular method selected from Southern blotting, Northern blotting, PCR, RT-PCR, qRT-PCR, microarray and sequencing. In other aspect, the invention relates to a kit for in vitro diagnosis of the insulin-related diseases, preferably the above-mentioned diseases, the kit comprising a biomarker which comprises at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA. The invention provides a kit for in vitro amplification of pancreatic β-cells, including at least one microRNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof. Brief Description of Figures
Figure 1 shows the effect of VP A of 0.75-3 mM on PI-MSC viability as % Control.
*** p<0,001 vs. control. Figure 2 shows the effect of 25mM of Glucose, ImM of VP A, and 25 mM of Glucose+ 1 mM of VP A on the β-cell differentiation at the end of days 10, 20 and 30. Histogram plots showing flow cytometric insulin + cell number (%, Fluorescence intensity] (A], a graphic showing the analysis results (B], and mean ±SEM values are provided. *p<0,05, **p<0,01, ***p<0,001 vs. control. Figure 3 shows the effect of experimental conditions applied to PI-MSCs for 20 days on mRNA production. A graphic representing the analysis results of mRNA levels (fold increase] (A] and mean ±SEM values (B] are provided. *p<0,05; **p<0,01 and ***p<0,001 vs. control.
Figure 4 shows microscopic photographs of the control group received complete medium (a] and the group received glucose (b] at the end of 20-day experiment period. The upper panel is magnified 4X, the middle panel is magnified 10X and the lower panel is magnified 2 OX. *shows the stem cell colony.
Figure 5 shows microscopic photographs of the group received VPA (a] and the group received VPA+glucose (b] at the end of 20-day experiment period. The upper panel is magnified 4X, the middle panel is magnified 10X and the lower panel is magnified 2 OX. *shows the stem cell colony and□ shows a β-cell like round cell.
Figure 6 shows the effect of experimental conditions applied to PI-MSCs for 20 days on ROS production. Dot plots showing flow cytometric DCF Fluorescence intensity (Fold Increase] (A], a graphic representing the analysis results (B] and mean ±SEM values are provided. **p<0,01 and ***p<0,001 vs. control, ##p<0,01 and ###p<0,001 vs. group received glucose.
Figure 7 shows insulin amounts (ng/^g protein] released from the cells as a result of 5.5 mM of glucose and 25 mM glucose administration. **p<0,01 and ***p<0,001 vs. control group.
Figure 8 demonstrates graphics showing OCT3/4, c-Myc and Nanog protein bands and their intensities in cytoplasmic (left panel] and nuclear (right panel] fractions of PI-MSCs. *p<0,05, **p<0,01, ***p<0,001 vs. control, ##p<0,01; ###p<0,001 vs. group received glucose, and +p<0,05 vs. group received valproic acid. A.U.: Arbitrary Unit
Figure 9 demonstrates graphics showing HDAC1 and HDAC3 protein bands and their intensities in cytoplasmic (left panel] and nuclear (rightpanel] fractions of PI-MSCs. **p<0,01, ***p<0,001 vs. control, ##p<0,01; ###p<0,001 vs. group received glucose. A.U.: Arbitrary Unit.
Figure 10 demonstrates graphics showing H4-K12 Ace, H3-K9 Ace and H3-K9 TriMe protein bands of PI-MSCs, and band intensities thereof. *p<0,05, **p<0,01, ***p<0,001 vs. control, #p<0,05 ##p<0,01; ###p<0,001 vs. group received glucose, +p<0,05 vs. VPA group. A.U.: Arbitrary Unit.
Figure 11 shows the changes in the expression levels of some miRNAs in the control group and the glucose-treated group. A statistically significant increase was found only in miRNAs that were above the blue line, while red circles show miRNAs, the gene expression levels of which are increased (p<0,05].
Figure 12 shows the changes in the expression levels of some miRNAs in the control group and the VPA-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased. A statistically significant increase was found only in miRNAs that were above the blue line (p<0,05).
Figure 13 shows the changes in the expression levels of some miRNAs in the control group and the VPA+glucose-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased. A statistically significant increase was found only in miRNAs that were above the blue line (p<0,05).
Figure 14 shows the changes in the expression levels of some miRNAs in the glucose-treated group and the VPA+glucose-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased. A statistically significant increase or decrease was found only in miRNAs that were above the blue line (p<0,05]. Figure 15 shows the changes in the expression levels of some miRNAs in the VPA+glucose-treated group vs. VPA-treated group. Red circles show miRNAs, the gene expression levels of which are increased, whereas green circles show miRNAs, the gene expression levels of which are decreased. Figure 16 shows the grade for gene expression level of miRNAs in each group. Gene expression levels increase from green to red.
Detailed Description of the Invention
In one aspect, the invention relates to at least one RNA selected from the group consisting of miR- 18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, for use in the treatment of the insulin-related diseases, preferably diabetes and obesity.
In another aspect, the present invention relates to use of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof in the treatment of the insulin-related diseases, preferably diabetes and obesity as a medicament in combination with antidiabetic/anti-obesity drugs.
In other aspects, the invention relates to use of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, alone or in combination with other antidiabetic, anti-obesity and anti-inflammatory drugs, in order to produce new β-cells in the cellular treatment of the insulin-related diseases, preferably diabetes and obesity. It is also suggested that more efficient cellular treatment methods can be developed by adding said molecules to the current β-cell differentiation protocols. In another aspect, the present invention relates to use of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, alone or in combination, or together with known biomarkers in the diagnosis of the insulin-related diseases, preferably diabetes and obesity. The term "mRNA" used in the present invention refers to messenger RNAs. The mRNA molecules transfer the genetic information obtained from DNA to the ribosome. The phrases "mRNA" and "messenger RNA" used in the present invention have the same meaning and are used interchangeably. The term "miRNA" used in the present invention refers to a small and non-coding RNA molecule. These molecules may be used for RNA silencing and regulation of post-transcriptional gene expression. The terms "micro RNA" and "miRNA" have the same meaning and may be used interchangeably. The term "IncRNA" used in the present invention refers to long non-coding RNAs. These RNAs are transcripts that do not code for protein and have a length of more than 200 nucleotides. The terms "lnc RNA" and "long non-coding RNA" used in the present invention have the same meaning and may be used interchangeably.
The term "diabetes mellitus" used in the present invention refers to type I diabetes mellitus and/or type II diabetes mellitus. The phrase "insulin-related diseases" used in the present invention refers to various neurodegenerative diseases such as lactose intolerance, fructose intolerance, galactosemia, glycogen storage disease, insulin resistance syndrome, syndrome X, retinopathy, nephropathy, foot ulcers, hypertension, hyperlipidemia, metabolic syndrome, gall bladder diseases, osteoarthritis, cardiovascular diseases, stroke, sleep apnea, liver disease, asthma, respiratory distress, menstruation disorders, musculoskeletal system disorders, skin diseases, polycystic ovarian syndrome, immune system disorders, diabetes mellitus, obesity and Alzheimer's disease and the other types of dementia, Parkinson's disease and other Parkinson's-related diseases, Huntington's disease, amyotrophic lateral sclerosis (ALS], Batten disease, motor neuron disease, spinal muscular atrophy, Prion disease, spinocerebellar ataxia, lewy body dementia, multiple sclerosis (MS], neuropathy and Friedrich ataxia.
MiR-184 is important for the regulation of β-cell mass increase i.e. compensation, in the case of insulin resistance. Ago2, which is the target of miR-184 in pancreatic islets, is part of the RNA- induced silencing complex required for targeting mRNAs. Loss of Ago2 blocks the compensatory increase of β-cells, which develops in response to insulin resistance by increasing the expression of miR-375 targets. Interestingly, miR-184 was suppressed in the islets of insulin resistant mice and humans, resulting in increased expression of Ago2, thereby compensatory increase of β-cells being suppressed. As a result of overexpression of miR-184 in ob/ob diabetic mice, Ago2 levels are decreased and compensatory increase of β-cells is suppressed.
MiR-335 is associated with β-cell function, rather than β-cell differentiation. In one study, islets were isolated from Goto Kakizaki (GK] rats which were used as a type 2 diabetes mellitus, a β-cell dysfunction. It has been determined that miR-335 expression is increased in these islets and that their targets are mRNAs of Stxbpl, Sytll and Snap25 which are molecules associated with insulin exocytosis (Esguerra et al., 2011]. In addition, overexpression of miR-335 leads to a decrease in glucose-induced insulin secretion and a decrease in depolarization-induced insulin exocytosis. In VPA-treated groups in which we detected β-cell differentiation, unresponsiveness to glucose- induced insulin secretion is parallel to the increase in miR-335 expression.
The relationship between Mir-433-5p and β-cell differentiation is notyet known. Gua et al. (2014] conducted a study comparing human placenta-derived mesenchymal stem cells in terms of epigenetic changes and in particular miRNA changes targeting pluripotency genes, in monolayer and three-dimensional spheroid culture conditions. As a result, they showed that the pluripotent properties of three-dimensional spheroid cells and their ability to differentiate into neuronal cells are much higher. In addition, in accordance with these changes they also determined that synthesis levels of certain miRNAs involved in maintaining the stem cell potency, among which is miR-433, are increased, and acetylation levels of H3-K9 are also increased in promoter regions of 0ct4, Sox2 and Nanog. On the basis of these results, they noted that spheroidal culture increases the potency of human mesenchymal stem cells and change the epigenetic state of pluripotent genes. Another study supporting the idea that miR-433 has a positive effect on potency, was performed by Kim et al. (2013]. In this study, it was determined that miR-433 mediates estrogen receptor-associated receptor gamma-suppressed differentiation in the C3H10T1/2 mesenchymal cell line. Interestingly, while an increase in expression of miR-433-5p is observed in all groups in which β-cell differentiation is detected in the studies of the present invention, this increase is statistically significant only in the VPA-treated group in which the highest differentiation level was observed.
MiR-30d is associated with pancreas development, whereas miR-19b is associated with HDCAs.
Members of the MiR-30 family are produced at high rates in the human fetal pancreas and are associated with epithelial-mesenchymal transition. These miRNAs inhibit mesenchymal mRNAs such as vimentin and Snaill and allow pancreatic mesenchymal stem cells to transform into insulin producing cells. It has been shown that miR-30d, a member of this family, regulates the β- cell transcription factor MafA, butdoes not affect Pdx-1 and NeuroDl. MiR-30d increased the MafA level and the transcription of the insulin gene.
MiR-19b has been reported to show an increase in acute myeloid leukemia cells treated with Vorinostat, a class I and II HDAC inhibitor. However, there was no study showing the relationship of miR-19b with HDAC except this study. It has also been shown that miR-19b is expressed at high levels in pancreatic progenitor cells and that this miRNA targets the 3 'UTR region of NeuroDl mRNA, thereby reducing the protein and mRNA levels of this transcription factor. It has also been shown that MiR- 19b inhibits insl expression in MIN6 cells, does not affect ins2, and has little effect on the proliferation of pancreatic progenitor cells. These results demonstrate that miR-19b can regulate β-cell differentiation and function by reducing the expression of Insl by targeting the transcription factor NeuroDl. In the present study, it was determined that glucose administration to PAC-MSCs in combination with VPA results in β-cell differentiation differently from VPA or glucose administration alone. MiR-503 was found to be significantly lower in serum of both diabetic and obese individuals than controls. On the other hand, it has been reported that the expression of CX3CL1 is regulated by suppression of miR-424 and miR-503 in response to microbial administration in epithelial cells of the HDAC and NF-κΒ signal. In the study of the present invention, a statistically significant decrease in VPA + glucose group in expression of miR- 503, but a statistically non-significant decrease was found in the cells treated with VPA alone.
MiR-124 is one of the best characterized and most abundantly expressed neuronal miRNAs. Overexpression of MiR-124 results in an increase in the expression of neuronal markers, and thus, neurite growth, indicative of neuronal differentiation, has also been shown to manifest itself as morphological changes. In some studies conducted in vertebrates, miR-124 has been identified as a stimulant for neuronal differentiation and as an inhibitor of self-renewal occurred in progenitor cells. However, it is not known how miR-124 directs neurogenesis in mesenchymal stem cells. In the study of the present invention, findings which suggest that miR-124 mediates differentiation of glucose-treated PI-MSCs into β-cells, were found for the first time in the literature.
Studies have shown that histone deacetylase inhibitor, trichostatin A, regulates the transcriptional activation of miR-146a by increasing the binding activity of NF-kB to DNA in macrophages. This finding suggests that miR-146a plays a role in the regulation of class I and II HDACs. There is no explicit study in the literature regarding the possible relationship of MiR-146a with β-cell differentiation. A number of studies have identified the presence of miR-146 circulating in the blood of diabetic individuals, suggesting that this miRNA may be a biomarker for the diagnosis of type 2 diabetes mellitus. In the study of the present invention, it was the first time in the literature that findings suggest that miR-146a mediates the differentiation of glucose-mediated PAC-MSCs into β-cells. In the study of the present invention, findings which suggest that miR- 146a mediates differentiation of glucose-treated PI-MSCs into β-cells, were found for the first time in the literature.
In another aspect, the invention relates to a method for the formation of pancreatic β-cell suitable for the treatment of insulin-related diseases, preferably diabetes and obesity, comprising in vitro administration of at least one RNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, to the stem cells.
In a further aspect, the invention relates to use of pancreatic β-cells obtained by co-administration of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof to the stem cells with at least one other antidiabetic, anti-obesity or anti-inflammatory agent, for the treatment of the insulin-related diseases, preferably for the treatment of diabetes and obesity. In other aspect, the invention relates to use of pancreatic β-cells obtained by in vitro administration of at least one RNA selected from the group consisting of miR-18a, miR-19b, miR- 30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, to the stem cells in combination with at least one other antidiabetic, anti-obesity or anti-inflammatory agent in the treatment of the insulin-related diseases, preferably in the treatment of diabetes and obesity.
The present invention also relates to a method for use in the diagnosis of the insulin-related diseases, preferably diabetes and obesity, comprising detecting the presence of at least one of biomarker comprising at least one RNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof, in all body fluids such as blood, plasma, serum, milk, bronchoalveolar fluid and cerebrospinal fluid.
Said molecules may be determined by precise molecular methods such as Southern Blot, Northern Blot, PCR, RT-PCR, qRT-PCR, microarray and sequencing and the methods that may be used are not limited to said techniques.
"Antidiabetic agent" used in the present invention means agents used in the treatment of diabetes mellitus, whereas "anti-obesity agent" refers to agents used in the treatment of obesity. All molecules (alone or in combination], all the related RNAs and DNAs, which were described in the present invention and detailed above can be used in cellular therapy in conjunction with antidiabetic, anti-obesity or anti-inflammatory drugs.
Said antidiabetic agents may be selected from insulin analogs, insulin sensitizers, insulin secretagogues, aldose reductase inhibitors, alpha glucosidase inhibitors, amylin analogs, peptide analogs, sodium glucose transporter 2 (SGLT] inhibitors and glucosuric agents. Insulin that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of insulin, insulin lispro, insulin aspart, insulin glulisine, insulin zinc, isophane insulin, insulin glargine, insulin detemir. Insulin sensitizers that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of metformin, phenformin, buformin, siglitazone, darlitazone, englitazone, lobeglitazone, netoglitazone, rivoglitazone, aleglitazar, saroglitazar, tesaglitazar, rosiglitazone, pioglitazone, and troglitazone. Insulin secretagogues that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of acetohexamide, carbutamide, chlorpropamide, metahexamide, tolbutamide, tolazamide, glibenclamide, glibornuride, glicetanyl, gliclazide, gliflumide, glipizide, gliquidone, glisoxepide, glyclopyramide, glimepiride, repaglinide, mitiglinide, exenatide, liraglutide, taspoglutide, albiglutide, lixisenatide, dulagutide, semaglutide, alogliptin, anagliptin, gemigliptin, linagliptin, omarigliptin, saxagliptin, sitagliptin, tenegliptin, vildagliptin, fasiglifam, and nateglinide.
Aldose reductase inhibitors that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of epalrestat, fidarestat, ranirestat, tolrestat, and zenarestat.
Alpha glucosidase inhibitors that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of miglitol, acarbose, and voglibose.
Amylin analog that can be used in combination with the molecules listed above in the context of the present invention is pramlintide.
Sodium glucose transporter 2 (SGLT] inhibitors that can be used in combination with the molecules listed above in the context of the present invention may be selected from the group consisting of canaglifozin, dapagliflozin, empagliflozin, remogliflozin, sergliflozin, and tofogliflozin.
In addition to the above, the antidiabetic agent may also be benfluorex or bromocriptine.
Said anti-obesity agents may be selected from the group consisting of 4-methyl amphetamine, amfecloral, amfepentorex, amfepramon, aminorex, amphetamine, atomoxetine, benfluorex, benzphetamine, bupropion, cathine, cathinone, chlorphentermine, cyclazindol, clobenzorex, cloforex, clominorex, clotermine, dexfenfluramine, dextroamphetamine, dexmethylphenidate, difemetorex, dimethylcathinone, difemethoxydine, ephedrine, ephedra, ethylamphetamine, etolorex, fenbutrazate, fencamfamin, fenethylline, fenfluramine, fenproporex, fludorex, fluminorex, furfenorex, indanorex, khat, levopropylhexedrine, lisdexamfetamine, manifaxine, mazindol, mefenorex, methamphetamine, methylphenidate, norfenfluramine, pemoline, pentorex, phendimetrazine, phenethylamine, phenmetrazine, phentermine, phenylpropanolamine, phsylorex, pipradrol, prolintane, propylhexedrine, pseudoephedrine, pyrovalerone, radafaxine, reboxetine, setazindol, sibutramine, synephrine, tesofensine, viloxazine, xylopropamine, zylofuramine, drinabant, ibipinabant, otenabant, rimonabant, rosonabant, surinabant, taranabant, 5-HTP, galactomannan, glucomannan, L-DOPA, L- phenylalanine, L-tryptophan, L-tyrosine, lorcaserin, Lu AA-33810, naltrexone, naloxone, oxyntomodulin, P57, peptide YY, topiramate, yohimbine, zonisamide, water, cetilistat, 2,4- dinitrophenol, dirlotapide, miltratapide, oleoyl estron, orlistat, simmondsin and sterculia, or multiple combinations thereof.
Said anti-inflammatory agents may be selected from the general groups consisting of pyrazolone/pyrazolidines, salicylates, acetic acid derivatives, oxicams, propionic acid derivatives, N-arylanthranilic acids, coxibs and the other agents.
Said anti-inflammatory agents may be selected from the groups consisting of aminophenazone, ampyrone, clophenazone, famprofazone, feprazone, kebuzone, metamizole, mofebutazone, morazone, nifenazone, oxyphenbutazone, phenazone, phenylbutazone, propyphenazone, sulfinpyrazone, suxibuzone, acetylsalicylic acid, aloxypyrine, benorilate, carbasalate calcium, diflunisal, ethenzamide, guacetical, magnesium salicylate, methyl salicylate, salsalate, salicylamide, salicylic acid, sodium salicylate, aceclofenac, acemetacin, alclofenac, amfenac, bendazac, bromfenac, bumadizone, bufexamac, diclofenac, difenpiramide, etodolac, felbinac, fenclozic acid, fentiazac, indomethacin, indomethacin farnesyl, isoxpac, ketorolac, lonazolac, acemetacin, prodolic acid, proglumetacin, sulindac, thioipinac, tolmetin, zomepirac, ampiroxicam, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, alminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, miroprofen, naproxen, oxaprozin, pirprofen, suprofen, tarenflurbil, tepoxalin, tiaprofenic acid, vedaprofen, naproxcinod, azapropazone, etofenamate, flufenamic acid, flunixine, meclofenamic acid, mefenamic acid, morniflumate, niflumic acid, tolfenamic acid, flutiazin, apricoxib, celecoxib, simicoxib, deracoxib, etoricoxib, firocoxib, lumiracoxib, mavacoxib, parecoxib, robenacoxib, rofecoxib, valdecoxib, aminopropionitrile, benzydamine, chondroitin sulphate, diacerein, fluproquazone, glucosamine, glycosaminoglycan, hyperforin, nabumetone, nimesulide, oxaceprol, proquazone, tenidap, and orgotein.
In a further aspect, the invention relates to pharmaceutical compositions comprising β-cells obtained by the methods according to the invention. Said pharmaceutical compositions may contain at least one other excipient in addition to the beta cells. The pharmaceutical compositions according to the invention may contain at least one other active ingredient in addition to the β- cells. The other active ingredient may be selected from the antidiabetic or anti-obesity agents described in detail above, or a combination thereof. The other active agent may be formulated together with β-cells, or it may be formulated separately and administered to the patient simultaneously, sequentially or at different times.
After the pharmaceutical compositions according to the present invention are administered to the culture medium in vitro to form beta cells, these cells can be injected intravenously. Formulations suitable for injection may be formulated using a sterile solvent or any pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be selected from sterile water, saline, or the existing cell culture media within the scope of state of the art, but is not limited thereto.
The pharmaceutical compositions according to the present invention may be administered parenterally in the form of an injectable formulation. Formulations suitable for injection may be formulated using a sterile solvent or any pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be selected from sterile water, saline, or the existing cell culture media within the scope of state of the art, but is not limited thereto. In a further aspect, the invention relates to a method of β-cell differentiation, comprising a step of obtaining β-cells by administrating a HDAC (Histone deacetylase] inhibitor and glucose to pancreatic mesenchymal stem cells. In a preferred embodiment of the invention valproic acid or a pharmaceutically acceptable salt thereof, such as sodium valproate is used as the HDAC inhibitor.
In another preferred embodiment of the invention, the HDAC inhibitor is used in a range of 0.5 mM to 5 mM, preferably in a range of 0.75 mM to 3 mM, particularly preferably in a range of 1 mM to 2 mM. In one embodiment of the invention, the β-cell differentiation protocol uses glucose in a range of 5 mM to 50 mM, preferably in a range of 10 nM to 40 mM, particularly preferably in a range of 20 mM to 30 mM. In another aspect, the invention relates to pharmaceutical formulations comprising at least one RNA selected from the group of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR- 335, miR-433-5p ve miR-503, or a DNA, mRNA or IncRNA thereof.
The pharmaceutical formulations according to the invention may, in addition to said RNAs, contain at least one pharmaceutically acceptable excipient and/or additional active ingredient
The other active agent mentioned herein may be an antidiabetic agent or an anti-obesity agent or anti-inflammatory agent Details of these agents are given in the context of the invention. The formulations according to the invention may be prepared in the form of any of the existing dosage forms known in the art of composition. Said dosage form may be administered to a patient orally, rectally, vaginally, intratumorally, subcutaneously, intracutaneously, intravenously, intracerebroventricularly, intramuscularly, intraarterially, intratracheally, interperitoneally, parenterally, topically, or by means of medical devices. The formulations according to the present invention can be suitably formulated to be targeted to, or to be carried by, the cells via nanoparticles, liposomes and the other similar carriers, or they can be administered by said routes. The formulations according to the present invention may be formulated in a manner suitable for administration to the patient by nasal, spray, oral, aerosol, rectal or vaginal route of administration, or they can be administered to the patient by said routes.
The pharmaceutical compositions according to the present invention may be administered parenterally in the form of an injectable formulation. Formulations suitable for injection may be formulated using a sterile solvent or any pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be selected from sterile water, saline, or the existing cell culture media within the scope of state of the art, but is not limited thereto.
Examples showing the principle of the invention and giving detailed information about the invention are given below. The examples are provided to provide a better understanding of the invention, and the scope of the invention is not limited in any way to these examples. Example 1: Materials and Methods Used in the Experimental Procedures
A) Cell Culture
The pancreatic islet-derived mesenchymal stem cells (PAK-15 MKH] used in this study were isolated from adult (2.5-3 months of age] Wistar albino rats and characterization studies were completed. All cell culture studies were carried out in Istanbul University, Faculty of Sciences, Department of Biology, Department of Molecular Biology, Primer and Stem Cell Research Laboratory. PI-MSCs between 6 and 9 passages were used in this study. Cells were grown in Minimum Essential Medium (MEM, Gibco, 21090-022] supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, 10500], an antibiotic mixture (100 units/ml penicillin, 100 μg/ml streptomycin, Gibco-15240-122] and glutamin (2mM, Gibco, 25030], called complete medium, at 37°C, 5% C02 and 95% air at a humidified incubator, known as standard culture conditions. Cells reproduced in sterile culture dishes were passaged after filling at least 80% of the culture dish surface. During this process, the cells were allowed to remove from the culture dish by applying 0,25% trypsin-EDTA (Sigma-T4174]. After the trypsin was inactivated by adding complete medium, the cells were collected by centrifuging at 1500 rpm for 5 minutes, and a plating process was performed in a new culture dish after cell counting.
B) Preparation of Experimental Conditions
Four different experimental conditions were applied to PI-MSCs. These are i] a complete medium which is a normal growing and developing medium, ii] a complete medium containing 25 mM of glucose, iii] a complete medium containing 1 mM of Valproic acid, and iv] a complete medium containing 1 mM of Valproic acid+25 mM of glucose. D-(+]-Glucose (Sigma G7021] was preferred to create glucose-containing conditions and sodium valproate (Sanofi Aventis, Depakine] was used for valproic acid-containing conditions.
Example 2: Determination of Cell Viability by MTT Test
MTT (3-(4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide] test is a toxicity test aimed at directly assessing cell viability and indirectly cell death. The basis of this method is the reduction of the tetrazolium ring of MTT to a blue-violet, water-insoluble formazan by succinate dehydrogenase, a mitochondrial enzyme, in viable cells. The capability of the cells to reduce MTT is accepted as a criterion of cell viability and formazan density obtained as a result of this reaction is directly proportional to the number of viable cell. Viable cells, the mitochondrial function of which is undistorted, are stained in purple color, whereas dead cells or cells having impaired mitochondrial function are not stained. After the formazan crystals formed by viable cells to which MTT is applied are dissolved with dimethylsulfoxide (DMSO], the reaction result is determined by the colorimetric measurement method.
For the MTT assay, 5,000 of PI-MSC were plated on 96-well culture plates and incubated for 24 hours under standard culture conditions for adhesion and proliferation of cells onto the surface of the culture dish. VPA at a dose of 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, selected in accordance with the literature was applied for 24 hours to determine non-toxic doses of VPA. (Haumaitre et al., 2008]. Cells grown in a complete medium were used as a control. 50 mg of MTT (Applichem, A2231 0001] were sterilized by passing through a filter of 0.22 μιη after bringing to 10 ml with MEM and dissolving. At the end of the experiment, MTT solution was added to each well in a volume of 30 μΐ for 100 μΐ of medium, and the culture plates were wrapped with aluminum foil and incubated under standard culture conditions for 4 hours. At the end of this period, the medium was removed from the wells in order to solubilize the formazan crystals and the culture plate was shaken on an orbital shaker at 250 rpm for 2 minutes, after adding 100 μΐ of DMSO to each well. After rinsing, the absorbance values were taken at 570-630 nm wavelength in a culture plate microplate reader (Biotek, Microquant]. The mean absorbance values of the cells in the control group were taken as 100% and the viability rate in the other groups was calculated as control%. The experiments were performed in triplicate and 10 wells were used for each group. Example 3: Detection of Cell Differentiation
A) Detection of Insulin-producing Cells
Demonstration of insulin, a specific marker of a mature β-cell, is essential for the detection of the final differentiation. For this purpose, insulin positive cells were detected by flow cytometry technique 10, 20 and 30 days after the substance administration. Cells were fixed by applying 2% paraformaldehyde at +4°C for 10 minutes and 10-minute permeabilization with PBS containing 0.05% triton X-100. Insulin primer antibody (Santa Cruz, sc-9168, 1:10] was then applied at room temperature for 30 minutes. After the cells were washed with PBS, fluorescein isothiocyanate (FITC] conjugated secondary antibody (Invitrogen-65-6111, 1 :10] was applied for 30 minutes in the dark and then washed. Analysis was performed on the FL-1 channel. The fluorescence intensity value of the control group was subtracted from the fluorescence intensity values of all groups using the BD Cell Quesht Pro program to calculate net fluorescence intensities.
B) Determination of Gene Expression Level of β-Cell-Specific Molecules
The gene expression levels of Pdx-1, Pax4/6, Nkx-2.2/6.1, Neurogenin-3, ins2 and Glut-2, which are transcription factors involved in the production of insulin and are other proteins specific for β-cells, were demonstrated by real-time reverse transcriptase polymerase chain reaction (qRT- PCR] method in order to determine β-cell differentiation. All procedures were performed using sterile equipment in a sterile environment The sterile cabinet and micropipettes were first cleaned with alcohol and then RNAse ZAP (Sigma, -R20203]. Cells were washed 2 times with cold PBS (1M, pH: 7.4]. 1 ml of cold Trizol (Invitrogen-15596-026) 1x106 cells was added to the eppendorf tubes. Each eppendorf tube was left in ice for 5 minutes. To all eppendorf tubes, 200 μΐ,, of chloroform cooled to +4°C was added and stirred for 15 seconds. After this, the Eppendorf tubes were left on ice for 5 minutes. The tubes were centrifuged at 12,000 rpm for 15 minutes at +4°C. The clear chloroform phase containing the nucleic acids was carefully collected and transferred to freshly prepared eppendorf tubes. To these eppendorf tubes was added isopropanol cooled to +4°C and the Eppendorf tubes were gently shaken. Eppendorf tubes were left on ice for 10 minutes and centrifuged at 12,000 rpm for 10 minutes at +4°C. 78% ethanol prepared in 1 ml of water with DEPC (Diethylpyrocarbonate, Sigma-D-5758] cooled to -20°C was added to the eppendorf tubes and centrifuged at 7600 rpm for 5 minutes at +4°C. Finally, 20 μΐ of water with DEPC was added to the Eppendorf tubes to allow total RNA to be dissolved. Total RNA level was measured in a Qubit Fluorimeter (Invitrogen] using the Qubit® RNA HS Assay Kit (Thermofisher, Q32852]. 1 μg of RNA was converted to cDNA by reverse transcriptase (NEB, ProtoScript First Strand cDNA Synthesis Kit, E6300S]. For each sample, a mixture of RNA and primer d(T]23 was first prepared. RNA denaturation was performed at 70°C for 5 minutes. After addition of the reaction mixture and the enzyme mixture to the tubes, incubation was carried out for 1 hour at 42°C followed by incubation at 80°C for 5 minutes in order to stop the enzyme activity. Thermal Cycler device (Techne] was used for these steps. Total cDNA level was measured in a Qubit Fluorimeter (Invitrogen] using the Qubit ssDNA Assay Kit. 300 ng of cDNA, gene expression assays consisting of Probe+Primers and TaqMan master mix (Applied Bioystems] were used for PCR and the products were obtained according to the instructions provided by the kit. Incubation of 2 minutes was performed at 50°C followed by denaturation step at 50°C for 2 minutes. 40 cycles consisting of an incubation step at 95°C for 15 seconds and at 60°C for 1 minutes were performed for the amplification. A 7500 Fast Real Time PCR System (Applied Biosystems] was used for this prosedure. The results were calculated as fold increase in 2ACT values.
C) Insulin Secretion Test
In order to test whether or not the differentiated β-cells were functional, the ability of these cells to release insulin in response to increase in glucose was measured. For this purpose, after 20 days of substance administration, the cells were incubated in DMEM medium containing 5.5 mM/L of glucose and 0.5% BSA for two hours subsequent to washing with PBS. After the culture medium was collected, the cells were washed with PBS and incubated for two hours in DMEM medium containing glucose (25 mM/L) at high concentration. Insulin levels of the collected media were determined by sandwich ELISA technique and the protocol provided by the kit (Millipore, EZRMI- 13K]. The total protein content of the cells was calculated by the Bradford test and a normalization process was performed proportioning the insulin levels to the protein levels.
Example 4: Flow Cytometric ROS Measurement with DCFDA
2',7'-dichlorofluorescein diacetate (DCFDA] is a fluorogenic agent that can readily pass through the cell membrane and allows levels of reactive oxygen species (ROS] such as hydroxyl and peroxyl in the cell to be measured. When DCFDA is introduced into the cell, the acetyl group is removed by some esterases and loses its ability to emit fluorescence. When it interacts with ROS in the cell, it oxidizes to 2',7'-dichlorofluorescein (DCF] and starts to emit fluorescence again. The resulting fluorescence intensity can be determined at maximum excitation (495nm] and maximum emission (529nm]. In this study, ROS level changes between experimental groups were determined by flow cytometry using DCFDA. Analyses were performed with 50,000 cells from each group and repeated at least 3 times. DCFDA (Sigma-D6883] was added to the cells at a concentration of 10 μΜ, incubated for 30 min in the dark at standard culture conditions and analyzed on FLl channel on a flow cytometer (BD, FACS Calibur]. The mean fluorescence intensity from the control group was taken as 1 and the ROS levels in the other groups were calculated as fold increase relative to the control group.
Example 5: Western Blot The lysis buffer (Invitrogen, FNN0011], to which the protease-phosphatase inhibitor mixture (Serva-39055] was added, was introduced to the cell pellet. This was allowed to stand for 30 minutes with stirring once every 10 minutes on ice. Ultrasonication was performed 5 times for 10 seconds. After centrifugation at 13000 rpm at °C for 10 minutes, the supernatant was stored in a freezer at -86°C for use as a cytoplasmic fraction. To the pellet was added 5 mM HEPES, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol (v/v], 300 mM NaCl; pH 7.9 was added, and a nuclear fraction was obtained by centrifuging at 24,000 g for 20 minutes (Abeam protocol book].
A) Preparation of Cell Lysates
The lysis buffer (Invitrogen, FNN0011], to which the protease-phosphatase inhibitor mixture (Serva-39055] was added, was introduced to the cell pellet. This was allowed to stand for 30 minutes with stirring once every 10 minutes on ice. Ultrasonication was performed 5 times for 10 seconds. After centrifugation at 13000 rpm at 4°C for 10 minutes, the supernatant was stored in a freezer at -86°C for use as a cytoplasmic fraction. To the pellet was added 5 mM HEPES, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol (v/v), and 300 mM NaCl; pH 7.9 were added, and a nuclear fraction was obtained by centrifuging at 24,000 g for 20 minutes (Abeam protocol book]. B) Determination of Protein Levels by Bradford Technique
Total protein amount of the resulting fractions was determined by Bradford method. 80, 40, 20, 10 and 8 μg/ml of Bovine Serum Albumin (BSA; Biorad Assay Standard II BSA-5 500- 0007] were used as standard. Measurement was performed in 96- well plates. Three replicates were run from each standard and sample. 160 μΐ were pipetted to the wells from 1 :50 diluted samples and standards. 40 μΐ of dye (Biorad Protein Assay Dye Reagent-500-0006] was added and stirred. Following a 5-minute incubation period, the absorbance was read in a microplate reader (Biotek- Microquant] at a wavelength of 595 nm. Protein concentrations ^g/ml] were calculated using the standard plot.
C) Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Materials used for the preparation of running and loading gels are provided in Table 1.
Table 1: Materials used for the preparation of SDS-PAGE gels, and amounts thereof
Figure imgf000021_0001
Samples containing 30 μg protein for the cytoplasmic fraction and 10 μg protein for the nuclear fraction were prepared in a volume of 10 μΐ, and denaturation process was carried out by leaving a total of 20 μΐ of the mixture obtained by adding 10 μΐ of laemmli buffer, in a dry block heater heated to 100°C (Techne Dri-Block DB-2D] for 5 minutes. After a brief centrifugation, the wells of the prepared gels were loaded. 25 mM Tris, 192 mM Glisin, 0,1% SDS; pH:8,3 were used a the running buffer. Proteins were run at 70 V for 30 minutes, 100 V for 45 minutes, and at 120 V and +4°C until the running process was terminated. D) Transfer of Proteins from Gel to a Membrane
Proteins separated by gel electrophoresis were transferred from the gel to a PVDF (Polyvinylidene fluoride] membrane by wet transfer. Membranes (Millipore-Immobilon-PSQ] with a pore diameter of 0.2 μιη were used for the transfer of proteins less than 20 kDa, while membranes with a pore diameter of 0.45 μιη (Millipore-Immobilon-P] were preferred for the transfer of proteins larger than 20 kDa. Transfer was carried out in Tris-Glycine SDS buffer containing 20% methanol for 12 hours at a current intensity of 80mA and + 4°C. Ponceau S staining solution was applied to the membrane for 5 min, and the efficiency of the transfer was checked by observing the band densities. After the bands were observed, 0.1 M NaOH was applied for 5 min to remove the Poncaue S stain from the membrane.
E) Labelling Specific Protein Bands
The membrane was incubated with a blocking solution prepared with 5% skimmed milk powder for 1 hour at room temperature in a shaker. Membranes were incubated overnight at +4°C with primer antibodies (Table 3.2] prepared with appropriate dilution rate after blocking. After this process, the membranes washed with the washing buffer TBS-T (95% TBS, 5% Twin-20] were treated with the secondary antibodies specific for HRP (horseradish peroxidase]-labeled primer antibody (Table 3.2] for 1 hour at room temperature. After the membranes were washed with the washing buffer, specific protein bands were visualized by Kodak GL1500 gel imaging system using Western Blotting Luminol Reagent (Cell Signaling Technology, 7003] for chemiluminescence substrate. Protein densities were analyzed with Kodak Molecular Imaging Systems software depending on the chemiluminescence ratio. Normalization was done by proportioning the band densities of the protein samples subjected to the density analysis to the density of β-actin protein for the proteins studied in the cytoplasmic fraction and to the density of the TFIIB protein for the proteins studied in the nuclear fraction.
Table 2: Antibodies used in Western Blotting, dilution rates for these antibodies, incubation conditions and periods
Figure imgf000022_0001
Rabbit anti-Histone Deacetylase 3 CST-2632 1:1000 Overnight,+4°C (HDAC3)
Mouse anti-HDACl (10E2) CST-5356 1:1000 Overnight,+4°C
Rabbit anti-Nanog, N-terminus Millipore- 1:500 Overnight,+4°C
AB5731
Rabbit anti-c-Myc CST-9402 1:1000 Overnight,+4°C
Rabbit anti-Oct3/4 (H-134) sc-9081 1:500 Overnight,+4°C
Mouse anti-S-actin sc-47778 1:500 Overnight,+4°C
Rabbit anti-TFIIB sc-225 1:500 Overnight,+4°C
Goat anti-rabbit IgG CST-7074 1:1000 Overnight,+4°C
Bovine anti-rabbit IgG sc-2370 1:2500 Overnight,+4°C
Horse anti-mouse IgG CST-7076 1:1000 Overnight,+4°C
Example 6: Determination of Gene Expression Levels of MiRNA
As a result of a comprehensive literature review during the planning phase of this study, 39 miRNAs that would be related to the study by identifying microRNAs (miRNAs] associated with pluripotency, early pancreatic development, end term pancreas maturation, Type 2 diabetes mellitus and HDACs have been determined. These are mir-375, let7a-l, miR-7al, miR7a2, Iet7a2- 1, let7d, let7f,-l mir-7, mir-16, mir-195, miR-30a, miR-30d, miR-142-5p, miR- 335, miR-26a, miR- 3545-3p, miR-206, miR-19b-l, miR-494, miR-503, miR-18a, miR- 92b, miR-342, miR-184, miR- 338, miR-124-1, miR-124a, miR-15b, miR-24-1, miR- 296, miR-134, miR-145, miR-148b, miR-187, miR-449a, miR-99b, miR-146a, miR- 181a-l, miR433-3p, miR433-5p. U6 was used as an internal control. Results were calculated in 2ACT values.
A) MicroRNA Isolation
QIAGEN miRNeasy Mini Kit protocol was used. 2.5x106 PI-MSC, which had been subjected to experimental conditions for 20 days, were taken up in 1.5 ml eppendorf tubes. 700 ul of QIAzol Lysis Reagent was added to all tubes and homogenized by thorough mixing. After 5 minutes at room temperature, 140 μΐ,, of chloroform was added and gently shaken for 15 seconds by inverting. The tubes were left at room temperature for 3 minutes and then centrifuged at 12,000 g at +4°C for 15 minutes. The supernatant was gently collected and transferred to new collection tubes without disturbing very fine intermediate phase and the bottom phase. 100% ethanol that is 1.5 times the volume of the obtained supernatant was added and mixed. 700 ul of this mixture was transferred to 2 ml of collection tubes with filters. Centrifugation was performed at 8000xg for 15 seconds. 700 ul of RWT buffer was added to the columns. Centrifugation was performed at 8000xg at room temperature for 15 seconds. 500 μΐ of RPE buffer was added and centrifuged at 8000xg at room temperature for 15 seconds. Again, 500 μΐ of RPE buffer was added and centrifuged at 8000xg at room temperature for 2 minutes. The column was placed in eppendorf tubes of 1.5 ml. 50 μΐ of Rnase-free water was added to the column. The tube was centrifuged at 8000xg for 1 minute after sealing. miRNAs were allowed to be transferred to the tube together with water.
B) Measurement of RNA Levels
The Qubit® RNA HS Assay Kit (Thermofisher, Q32852] was used to quantify the amount of RNAs obtained as a result of isolation. Tubes of 0.5 ml were prepared for the standards and samples. Solution required for the samples and standards being studied by diluting Qubit RNA HS Reagent in Qubit RNA HS Buffer at a ratio of 1 :200. 190 μΐ and 199 μΐ of this solution were transferred to the standard tubes and the sample tubes, respectively. RNA levels were determined by Qubit Fluorimeter 2.0 (Invitrogen] after 10 μΐ of standards and 1 μΐ of samples were added, followed by incubation at room temperature for 5 min in the dark.
C) cDNA Extraction
Approximately 1 μg of RNA was converted to cDNA using reverse transcriptase enzyme (Qiagene, 2180 3]. For each sample, a total of 20 μΐ of mixture was formed. This mixture was composed of 4 μΐ of 5x miScript buffer, 2 μΐ of lOx miScript mix, 2 μΐ of miScript Reverse Transcriptase, 5.75 μΐ of RNase-free water, and 6.25 μΐ of RNA. For cDNA extraction, incubation was carried out at 95°C for 5 minutes to inactivate the reverse transcriptase enzyme after incubation at 37°C for 60 minutes. Thermal Cycler device (Techne] was used for these steps. D) qRT-PCR
100 μΐ tubes and QIAGEN miScript SYBR Green PCR Kit were used. The reaction mixture prepared in a total volume of 20 μΐ was composed of 4.5 μΐ of RNase-free water (QIAGEN], 10 μΐ of SYBR green (2xQuaintiTect SYBR Green PCR], 2 μΐ of master mix (lOx miScript Universal Primer], 2 μΐ of primer mix QIAGEN, miscript primer Assays] and 1.5 μΐ of cDNA. A reaction mixture of 1.5 μΐ without Rnase-free water was used as a negative control instead of cDNA. U6 miRNA was used as an internal control. qRT-PCR analysis was performed using the Rotor-Gene Q Series Software 2.3.1 (Qiagen] program. Denaturation at 95°C for 15 minutes, 40 cycles of 94°C for 15 seconds, 55°C for 30 seconds and 70°C for 30 seconds were used as an amplification program. The CT values obtained were analyzed using the RT2 Profiler PCR Array Data Analysis (Qiagen] program. Example 7: Results of Cell Viability Test
Valproic acid was applied to PI-MSCs at a dose of 0.75-3 mM for 24 hours. The effect of valproic acid on cell viability at the specified dose range was determined by MTT technique and the results are shown in Figure 1.
Cell viability values (Control %] are as follows: 100±0.00% in the control group; 131.40±1.53 in the group treated with 0.75 mM of VP A; 107.50±1.76 in the group treated with 1 mM of VP A; 91.58±2.52 in the group treated with 1.5 mM of VP A; 76.48±2.12 in the group treated with 2 mM of VP A; 58.28±2.08 in the group treated with 2.5 mM of VP A; and 51.63±2.17 in the group treated with 3 mM of VPA. There was found a significant increase in the number of the cells only in the group treated with 0.75 mM of VPA versus control group. A significant decrease in cell viability was observed in VPA concentrations increased after the group treated with 1 mM of VPA. From these results, it was decided to continue with β-cell differentiation studies by applying 0.75 mM and 1 mM of VPA that are doses increasing or decreasing the cell viability.
Example 8: Results for β-Cell Differentiation
After applying VPA to PI-MSCs at a concentration of 0.75 and 1 mM for 10 days, the cells were labeled with insulin antibody and the number of positive cells was determined by flow cytometry. As a result, it was determined that both dose application stimulated β-cell differentiation but 1 mM of VPA led to relatively more differentiation. It was noted that 1 mM ofVPA resulted in 28.46% β-cell differentiation whereas 0.75 mM of VPA stimulated 26.25% differentiation. It was deemed appropriate to continue the studies with valproic acid at a concentration of 1 mM in which β-cell differentiation was stimulated by maintaining the cell viability.
The insulin + cell ratios recorded after administration of complete media, 25 mM Glucose, 1 mM VPA, and 1 mM VPA + 25 mM Glucose to PI-MSCs for 10, 20 and 30 days are shown in Figure 2. At the end of day 10, a significant β-cell differentiation was observed for the group receiving only 1 mM VPA. At the end of days 20 and 30, a significant β-cell differentiation was observed for the groups receiving 25 mM Glucose, 1 mM VPA and 1 mM VPA+25mM Glucose. It was found that the highest β-cell differentiation occurred in the groups for which the application was performed for 20 days. From these results, it was decided for the study to be continued with a 20-day experiment period.
A) Results for β-CeII Differentiation at the Level of Gene Expression Complete medium, 1 mM VPA, 25 mM Glucose and 1 mM VPA+25 mM Glucose was applied to PI- MSCs for 20 days, with the media being changed daily. At the end of the experiment period, RNA isolation of cells belonging to all groups was performed. cDNA was obtained and gene expression levels of ins2, Pdxl, Ngn3, Nkx6.1, Pax4, Pax6, Glut2 and ACTB were determined by qRT- PCR. Results are shown in Figure 3.
It was determined that the expression levels of the molecules involved in maintaining the β-cell differentiation and functions in the glucose-treated group were similar to the control group (p>0,05).
When VPA group was compared to the control group, a significant increase in ins2 and Pdx-1 gene products (*p<0,05] and also in NKX6.1 gene product (***p<0,001], and a significant decrease in Pax6 and Glut-2 gene products (**p<0,01] were determined.
When VPA+glucose-treated group was compared to the control group, a significant increase in ins2, Pdx-1, Ngn3, Pax4 and Glut-2 gene products (***p<0,001] and also in NKX6.1 gene product (**p<0,01], and a significant decrease in Pax6 gene product (***p<0,001] were determined.
B) Microscopic Results
Figures 4 and 5 show photographs taken at the end of 20 days on the inverted light microscope. The morphological similarity between the glucose-treated PI-MSCs and the complete medium is remarkable. The common characteristic of both groups is the formation of stem cell colonies of largely varying sizes (Figure 4]. This image corresponds to the characteristic of pluripotent cell. Conversely, the presence of round cells concentrated in colony-like structures in the PI-MSCs treated with VPA and VPA + glucose is noteworthy. It has been observed that the PI-MSCs surrounding the colonies of such cells have decreased in number such that the surrounding of the colonies is emptied and generally, the round cells separated from these colonies are spread to these spaces. It is noteworthy that the transition of round cells that have been separated, from an adherent form to a suspended form (Figure 5]. In this regard, it was found that the cells forming the colonies in the VPA and VPA + glucose-treated groups were round like the pancreatic islet cells.
C) Results for Changes in the Production of Reactive Oxygen Species
Changes in the levels of reactive oxygen species (ROS] which are formed as a result of an increase in the oxidative phosphorylation, one of the important characteristics of the differentiating cells, have been identified. At the end of the 20-day experiment period, ROS levels were measured by flow cytometry by administering to the cells DCFH-DA which is used as an indicator for ROS. The results were calculated as fold increase as compared to the control group (Figure 6). When the results were compared, a significant increase in all other groups versus the control group, and a significant increase in the VPA and VPA + G groups versus glucose group were found. D) Results for Insulin Secretion
Atthe end of day 20, cells from all experimental groups were subjected to an insulin secretion test to measure their ability to release insulin in response to glucose increase. A glucose concentration of 5.5 mM was applied to the cells as low glucose condition, whereas a glucose concentration of 25 mM was applied as high glucose condition. The results obtained for each group were normalized by proportioning to the total protein levels (Figure 7).
When glucose was applied at a concentration of 5.5 mM, insulin release was as follows: 0.15±0.01 ng^g protein in the control group; 0.29±0.05 ng^g protein in VPA-treated group; and 0.28±0.01 ng^g protein in VPA+glucose-treated group. When the groups were compared with each other, a significantly increased level of insulin secretion was observed in the VPA and VPA + glucose- treated groups compared to the control group (p<0,01].
When glucose was applied at a concentration of 25 mM, insulin release was as follows: 0.15±0.01 ng^g protein in the control group; 0.28±0.01 ng^g protein in the glucose group; 0.28±0.01 ng^g protein in VPA-treated group; and 0.30±0.01 ng^g protein in VPA+glucose-treated group. When the groups were compared with each other, a significantly increased level of insulin secretion was observed in all groups compared to the control group (p<0,01 for G and VPA; p<0,001 for VPA+glucose]. Example 9: Results for Changes in Pluripotent Character
Complete medium, 1 mM VPA, 25 mM Glucose and 1 mM VPA+25 mM Glucose was applied to PI- MSCs for 20 days, with the media being changed daily. At the end of the experiment period, cytoplasmic and nuclear fractions were obtained from cell lysates and 0ct3 /4, c-Myc and Nanog levels were determined by western blot method. The obtained optical density values were normalized by proportioning to the β-actin density for the cytoplasmic fraction and to the TF-IIB density for the nuclear fraction (Figure 8).
Amount of 0CT3/4 in the cytoplasm was found to be 1.17±0.06 for the control group; 0.69±0.18 for glucose-treated group; 0.32±0.10 for VPA-treated group; and 0.37±0.09 for VPA+G-treated group. Nuclear 0CT3/4 ratios were found to be 0.91±0.16 for the control group; 0.45±0.078 for glucose-treated group; 0.34±0.07 for VPA-treated group; and 0.37±0.02 or VPA+G-treated group.
When 0CT3/4 changes in the cytoplasmic fraction were considered, a significant decrease was observed in all groups compared to the control group (p<0,05 for G-treated group, p<0,01 for VP A- and VPA+G-treated group]. When 0CT3 /4 levels in the nuclear fraction were compared, there was a significant decrease in all groups compared to the control group (p<0,05].
Amount of cytoplasmic c-Myc were calculated as 1.07±0.04 for the control group; 0.60±0.015 for glucose-treated group; 0.36±0.06 for VPA-treated group; and 0.52±0.06 for VPA+G-treated group. Ratios of nuclear c-Myc were found to be 0.30±0.06 for the control group; 0.48±0.09 for glucose- treated group; 0.11±0.07 for VPA-treated group; and 0.07±0.01 for VPA+G-treated group. When cytoplasmic c-myc levels were compared, a significant decrease was found in all groups compared to the control group (p<0,001]. When the glucose-treated group and the VPA-treated group were compared, a significant decrease in c-myc levels was detected in the VPA-treated group compared to the glucose-treated group (p<0,01]. When VPA-treated group and VPA + G-treated group was compared, a significant decrease in c-myc levels was observed in VPA-treated group compared to the VPA + G-treated group (p<0,05]. When c-myc levels in the nuclear fraction were compared to the control group, a significant increase was found in glucose-treated group (p<0,05]. When the control group was compared to VPA- and VPA+G-treated group, a significant decrease was observed in VPA- and VPA+G-treated group (p<0,01]. A significant decrease was found in VPA- and VPA+G-treated group compared to glucose-treated group (p<0,001].
The amount of cytoplasmic Nanog was 1.32±0.21 for the control group; 0.74±0.07 for glucose- treated group; 0.1±0.01 for VPA-treated group; and 0.12±0.01 for VPA + G-treated group. Ratios of nuclear Nanog were 1.27±0.24 for the control group; 0.76±0.07 for glucose-treated group; 0.19±0.01 for VPA-treated group; and 0.13±0.01 for VPA + G-treated group. When the groups were compared with each other in terms of cytoplasmic Nanog levels, all groups showed a significant decrease compared to the control group (p<0,05 for G-treated group, p<0,001 for VPA- and VPA+G-treated groups]. Similarly, for levels of Nanog in the nuclear fraction a significant decrease was found in all groups compared to the control group (p<0,05 for G-treated group, p<0.001 for VPA- and VPA+G-treated groups]. A significant decrease was observed in VPA-treated group compared to glucose-treated group (p<0,05]. A significant decrease was observed in VPA+G- treated group compared to G-treated group (p<0,001]. Example 10: Results for Changes Occurred in Some of Class I HDACs and Histone Modifications
Complete medium, 1 mM VP A, 25 mM Glucose and 1 mM VPA+25 mM Glucose was applied to PI- MSCs for 20 days, with the media being changed daily. Subsequently, cytoplasmic and nuclear fractions were obtained from cell lysates and levels of HDACl and 3, H3K9-Ace, H3K9-Met, H4K12-Ace were determined by western blot method. Optical density values of the protein bands were normalized by proportioning to the β-actin density for the cytoplasmic fraction and to the TF-IIB density for the nuclear fraction.
Figure 9 shows the changes in cytoplasmic and nuclear fraction levels of HDACl and 3 among Class I HDACs. Ratios of cytoplasmic HDACl were 0.14±0.01 for the control group; 0.12±0.02 for glucose-treated group; 0.04±0.01 for VPA-treated group; and 0.03±0.01 for VPA + G-treated group. When the results were compared, a significant decrease was found in VPA- and VPA + G- treated groups compared to the control group (p<0,001]. When VPA- and VPA + G-treated groups were compared to glucose-treated group, a significant decrease was detected (p<0,001]. Ratios of nuclear HDACl were 0.43±0.12 for the control group; 0.31±0.07 for glucose-treated group; 0.09±0.05 for VPA-treated group; and 0.07±0.02 for VPA + G-treated group. When the results were compared, a significant decrease was detected in VPA- and VPA + G-treated groups compared to the control group (p<0,001]. When VPA- and VPA + G-treated groups were compared to glucose- treated group, a significant decrease was found (p<0,01].
Ratios of cytoplasmic HDAC3 were 0.15±0.03 for the control group0.12±0.02for glucose-treated group; 0.05±0.01 for VPA-treated group; and 0.04±0.00 for VPA + G-treated group. When the results are compared, a significant decrease was observed in VPA- and VPA+G-treated groups compared to glucose-treated group (p<0,001]. When VPA-treated group was compared to glucose-treated group, a significant decrease was found (p<0,01]. When VPA+G-treated group was compared to glucose-treated group, a significant decrease was found (p<0,001]. Ratios of nuclear HDAC3 were 0.14±0.01 for the control group; 0.11±0.07 for glucose-treated group; 0.03±0.00 for VPA-treated group; and 0.03±0.00 for VPA + G-treated group. A significant decrease was observed in VPA- and VPA+G-treated groups compared to the control group (p<0,01]. When VPA- and VPA+G-treated groups were compared to glucose-treated group, a significant decrease was observed (p<0,01]. Figure 10 shows the results of histone modifications. H4K12-Ace ratios were calculated as 0.006±0.001 in the control group; 0.005±0.001 in VPA-treated group; and 0.075±0.019 in VPA+G- treated group. When the results were compared, a significant increase was observed in VPA- treated group compared to the control and glucose-treated group (p<0,01]. When VPA+G-treated group was compared to the control and glucose-treated groups, a significant increase was observed (p<0,05). No significant difference was found between VPA- treated group and VPA+G- treated group (p>0,05).
H3K9-Met levels were calculated as 0.225±0.001 in the control group, 0.160±0.283 in glucose- treated group, 0.070±0.001 in VPA-treated group; and 0.080±0.042 in VPA + G-treated group. A significant decrease was observed only in VPA- and VPA+G-treated groups compared to the control group (p<0,05).
H3K9-Ace levels were calculated as 0.076±0.004in the control group, 0.108± O.OOlin glucose- treated group, 0.171±0.001 in VPA-treated group; and 0.119±0.130 in VPA + G-treated group. A significant increase was observed in VPA-treated group compared to the control and glucose- treated group (p<0,001]. A significant decrease (p <0.05] was found when VPA+G-treated group was compared with VPA-treated group, and a significant increase (p<0,05] was found when VPA+G-treated group was compared with the control group.
Example 11: Results for Differences in the Gene Expression Levels of Certain MiRNAs
Expression changes of 39 miRNAs were identified by qRT-PCR as a result of 20-day experimental conditions. All results were calculated using 2ACT (Table 3). Accordingly, changes in miRNA production were demonstrated between the groups (Figure 11, Figure 12 and Figure 13, Figure 14, Figure 15, Figure 16).
Table 3: 2ACT values of certain miRNAs in all experimental groups
Figure imgf000031_0001
When the results shown in Figure 11 were compared, a significant increase in the expression levels of miR-124 and miR-146a was observed in the glucose-treated group compared to the control group (p<0.05]. Remarkable but not significant increases were noted in miR-7, miR-184 and miR433-5p (p>0,05).
When the results shown in Figure 12 were compared, a significant increase in the products miR- 335, miR- 18a, miR-184 and miR433-5p was observed in VPA-treated group compared to the control group (p<0.05]. Remarkable but not significant increases were found for miR-7, miR-124, miR- 124a, miR-15b, miR- 134, miR-187 and miR433-3p(p>0,05]. When the results shown in Figure 13 were compared, a significant increase in the expression levels of miR-30d and miR-19b was observed in VPA+G-treated group compared to the control group (p<0.05]. Remarkable but not significant increases were found for miR-7, miR-335, miR- 3545, miR-18a, miR-184, miR-124a, miR-15b, miR-134, miR-187, miR-449, miR-99b, miR-433- 3p, miR433-5p, miR-146a (p>0,05]. It was determined that the expression of MiR-503 was decreased markedly, but this decrease was not statistically significant
When the results shown in Figure 14 were compared, a significant increase in the expression level of miR-30d was observed in VPA+G-treated group compared to G-treated group, whereas a significant decrease was observed in miR124 (p<0,05]. Remarkable but not significant increases were found for miR-335, miR-3545, miR-19b, miR-18a, miR-184, miR-338, miR-187, miR-99b, miR-433-5p. A significant decrease was observed in the expression level of MiR-503 (p<0,05].
When the results shown in Figure 15 were compared, no significant change in miRNA product was detected in VPA + glucose-treated group compared to the VPA group (p>0.05]. Remarkable but not significant increases were found for miR-30d, miR-187, miR-99b, miR-433-3p, miR-146. Remarkable but not significant decreases were found for miR-375 and miR-124.

Claims

At least one RNA molecule selected from the group consisting of miR-18a, miR-19b, miR- 30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA molecule thereof, for use in the treatment of an insulin-related disease.
A molecule for use according to Claim 1, wherein said insulin-related disease is selected from the group consisting of prediabetes, Type I diabetes mellitus, Type II diabetes mellitus, metabolic syndrome, obesity, lactose intolerance, fructose intolerance, galactosemia, glycogen storage disease, insulin resistance syndrome, syndrome X, retinopathy, neuropathy, nephropathy, foot ulcers, hypertension, hyperlipidemia, metabolic syndrome, gallbladder diseases, osteoarthritis, cardiovascular diseases, stroke, sleep apnea, liver disease, asthma, respiratory distress, menstruation disorders, musculoskeletal system disorders, skin diseases, polycystic ovarian syndrome, immune system disorders, Alzheimer's disease and the other types of dementia, Parkinson's disease and other Parkinson's-related diseases, Huntington's disease, amyotrophic lateral sclerosis (ALS], Batten disease, motor neuron disease, spinal muscular atrophy, Prion disease, spinocerebellar ataxia, lewy body dementia, multiple sclerosis (MS], neuropathy and friedrich ataxia.
A molecule for use according to Claim 1, wherein said molecule is provided in combination with at least one additional active substance selected from antidiabetic, anti-obesity and anti-inflammatory agents.
A molecule for use according to Claim 3, wherein said antidiabetic agent is selected from the group consisting of insulin analogs, insulin sensitizers, insulin secretagogues, aldose reductase inhibitors, alpha glucosidase inhibitors, amylin analogs, peptide analogs, sodium glucose transporter 2 (SGLT] inhibitors and glucosuric agents.
A molecule for use according to Claim 3, wherein the anti-obesity agent is selected from the group consisting of 4-methyl amphetamine, amfecloral, amfepentorex, amfepramon, aminorex, amphetamine, atomoxetine, benfluorex, benzphetamine, bupropion, cathine, cathinone, chlorphentermine, cyclazindol, clobenzorex, cloforex, clominorex, clotermine, dexfenfluramine, dextroamphetamine, dexmethylphenidate, difemetorex, dimethylcathinone, difemethoxydine, ephedrine, ephedra, ethylamphetamine, etolorex, fenbutrazate, fencamfamin, fenethylline, fenfluramine, fenproporex, fludorex, fluminorex, furfenorex, indanorex, khat, levopropylhexedrine, lisdexamfetamine, manifaxine, mazindol, mefenorex, methamphetamine, methylphenidate, norfenfluramine, pemoline, pentorex, phendimetrazine, phenethylamine, phenmetrazine, phentermine, phenylpropanolamine, phsylorex, pipradrol, prolintane, propylhexedrine, pseudoephedrine, pyrovalerone, radafaxine, reboxetine, setazindol, sibutramine, synephrine, tesofensine, viloxazine, xylopropamine, zylofuramine, drinabant, ibipinabant, otenabant, rimonabant, rosonabant, surinabant, taranabant, 5-HTP, galactomannan, glucomannan, L-DOPA, L-phenylalanine, L- tryptophan, L-tyrosine, lorcaserin, Lu AA-33810, naltrexone, naloxone, oxyntomodulin, P57, peptide YY, topiramate, yohimbine, zonisamide, cetilistat, 2,4-dinitrophenol, dirlotapide, miltratapide, oleoyl estron, orlistat, simmondsin and sterculia.
A molecule for use according to Claim 3, wherein the anti-inflammatory agent is selected from the group consisting of pyrazolone/pyrazolidines, salicylates, acetic acid derivatives, oxicams, propionic acid derivatives, N-arylanthranilic acids and coxibs.
A molecule for use according to Claim 1, wherein said molecule is provided in a pharmaceutical formulation.
A molecule for use according to Claim 7, wherein said pharmaceutical formulation is in a dosage form that may be administered to a patient orally, rectally, vaginally, intratumorally, subcutaneously, intracutaneously, intravenously, intracerebroventricularly, intramuscularly, intra-arterially, intrathecally, intranasally, interperitoneally, parenterally, topically, or by means of medical devices.
A molecule for use according to Claim 7, wherein said formulation is suitable to be targeted to, or to be carried by, the cells via nanoparticles, liposomes or other carriers.
A method for obtaining pancreatic β-cells in vitro, comprising treatment of mesenchymal stem cells with at least one microRNA selected from miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or a related IncRNA thereof.
A method according to Claim 10, wherein said method comprises treatment of mesenchymal stem cells with a HDAC (Histone deacetylase] inhibitor and/or glucose.
12. A method according to Claim 11, wherein said HDAC inhibitor is valproic acid or a pharmaceutically acceptable salt thereof.
13. A method according to Claim 11 or 12, wherein said HDAC inhibitor has a concentration ranging between 0.5 mM to 5 mM.
14. A method according to Claim 11, wherein said glucose has a concentration ranging between 5 mM to 50 mM.
15. Pancreatic β-cells obtainable by the method according to claim 15 for use in the treatment of an insulin-related disease.
16. β-cells for use according to Claim 15, wherein said pancreatic β-cells are provided in combination with at least one microRNA selected from miR-18a, miR-19b, miR-30d, miR-
124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or one or more related IncRNA thereof.
17. β-cells for use according to Claim 15, wherein said β-cells are provided in combination with at least one additional agent selected from antidiabetic, anti-obesity and anti-inflammatory agents.
18. β-cells for use according to Claim 15, wherein the insulin-related disease is selected from the group consisting of prediabetes, Type I diabetes mellitus, Type II diabetes mellitus, metabolic syndrome, obesity, lactose intolerance, fructose intolerance, galactosemia, glycogen storage disease, insulin resistance syndrome, syndrome X, retinopathy, neuropathy, nephropathy, foot ulcers, hypertension, hyperlipidemia, metabolic syndrome, gallbladder diseases, certain types of cancer, osteoarthritis, cardiovascular diseases, stroke, sleep apnea, liver disease, asthma, respiratory distress, menstruation disorders, musculoskeletal system disorders, skin diseases, polycystic ovarian syndrome, immune system disorders, Alzheimer's disease and the other types of dementia, Parkinson's disease and other Parkinson's-related diseases, Huntington's disease, amyotrophic lateral sclerosis (ALS], Batten disease, motor neuron disease, spinal muscular atrophy, Prion disease, spinocerebellar ataxia, lewy body dementia, multiple sclerosis (MS], neuropathy and Friedrich ataxia.
19. β-cells for use according to Claim 17, wherein said antidiabetic agent is selected from the group consisting of insulin, insulin lispro, insulin aspart, insulin glulisine, insulin zinc, isophane insulin, insulin glargine, insulin detemir, metformin, phenformin, buformin, siglitazone, darlitazone, englitazone, lobeglitazone, netoglitazone, rivoglitazone, aleglitazar, saroglitazar, tesaglitazar, rosiglitazone, pioglitazone, troglitazone, acetohexamide, carbutamide, chlorpropamide, metahexamide, tolbutamide, tolazamide, glibenclamide, glibornuride, glicetanyl, gliclazide, gliflumide, glipizide, gliquidone, glisoxepide, glyclopyramide, glimepiride, repaglinide, mitiglinide, exenatide, liraglutide, taspoglutide, albiglutide, lixisenatide, dulagutide, semaglutide, alogliptin, anagliptin, gemigliptin, linagliptin, omarigliptin, saxagliptin, sitagliptin, tenegliptin, vildagliptin, fasiglifam, nateglinide, epalrestat, fidarestat, ranirestat, tolrestat, zenarestat, miglitol, acarbose, voglibose, pramlintide, canaglifozin, dapagliflozin, empagliflozin, remogliflozin, sergliflozin, tofogliflozin, benfluorex and bromocriptine.
β-cells for use according to Claim 17, characterized in that said anti-obesity agent is selected from the group consisting of 4-methyl amphetamine, amfecloral, amfepentorex, amfepramon, aminorex, amphetamine, atomoxetine, benfluorex, benzphetamine, bupropion, cathine, cathinone, chlorphentermine, cyclazindol, clobenzorex, cloforex, clominorex, clotermine, dexfenfluramine, dextroamphetamine, dexmethylphenidate, difemetorex, dimethylcathinone, difemethoxydine, ephedrine, ephedra, ethylamphetamine, etolorex, fenbutrazate, fencamfamin, fenethylline, fenfluramine, fenproporex, fludorex, fluminorex, furfenorex, indanorex, khat, levopropylhexedrine, lisdexamfetamine, manifaxine, mazindol, mefenorex, methamphetamine, methylphenidate, norfenfluramine, pemoline, pentorex, phendimetrazine, phenethylamine, phenmetrazine, phentermine, phenylpropanolamine, phsylorex, pipradrol, prolintane, propylhexedrine, pseudoephedrine, pyrovalerone, radafaxine, reboxetine, setazindol, sibutramine, synephrine, tesofensine, viloxazine, xylopropamine, zylofuramine, drinabant, ibipinabant, otenabant, rimonabant, rosonabant, surinabant, taranabant, 5-HTP, galactomannan, glucomannan, L-DOPA, L-phenylalanine, L-tryptophan, L-tyrosine, lorcaserin, Lu AA-33810, naltrexone, naloxone, oxyntomodulin, P57, peptide YY, topiramate, yohimbine, zonisamide, cetilistat, 2,4-dinitrophenol, dirlotapide, miltratapide, oleoyl estron, orlistat, simmondsin and sterculia.
β-cells for use according to Claim 17, wherein said anti-inflammatory agent is selected from the group consisting of pyrazolone/pyrazolidines, salicylates, acetic acid derivatives, oxicams, propionic acid derivatives, N-arylanthranilic acids and coxibs.
22. β-cells for use according to Claim 15, wherein said β-cells are provided in a pharmaceutical composition comprising at least one excipient
23. β-cells for use according to Claim 22, wherein said pharmaceutical composition is suitable to be targeted to, or to be carried by, the cells via carriers selected from heparin, lactic acid based polymers, polyesters, hydrogels, biopolymer films, extravascular compartments, intravascular compartments, alginate, poly(hydroxyethylmethacrylate-methyl methacrylate], agarose, acrylonitrile copolimers, chitosan, and PEG nanoparticles and liposomes.
24. A method for in vitro diagnosis of the insulin-related diseases, wherein the method comprises use of at least one micro RNA selected from the group consisting of miR- 18a, miR- 19b, miR-30d, miR- 124, miR-146a, miR- 184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or one or more related IncRNA as a biomarker.
25. A method according to Claim 24, wherein said insulin-related disease is selected from the group consisting of prediabetes, Type 1 or Type II diabetes mellitus, metabolic syndrome, obesity, lactose intolerance, fructose intolerance, galactosemia, glycogen storage disease, insulin resistance syndrome, syndrome X, retinopathy, nephropathy, foot ulcers, hypertension, hyperlipidemia, metabolic syndrome, gall bladder diseases, osteoarthritis, cardiovascular diseases, stroke, sleep apnea, liver disease, asthma, respiratory distress, menstruation disorders, musculoskeletal system disorders, skin diseases, polycystic ovarian syndrome, immune system disorders, diabetes, obesity and Alzheimer's disease and the other types of dementia, Parkinson's disease and other Parkinson's-related diseases, Huntington's disease, amyotrophic lateral sclerosis (ALS], Batten disease, motor neuron disease, spinal muscular atrophy, Prion disease, spinocerebellar ataxia, lewy body dementia, multiple sclerosis (MS], neuropathy and Friedrich ataxia.
26. A method according to Claim 24, wherein said method comprises detecting one of said biomarkers in a sample selected from the group consisting of blood, plasma, serum, milk, bronchoalveolar fluid and cerebrospinal fluid.
27. A method according to Claim 24, wherein the method comprises detecting said biomarkers by a molecular method selected from Southern blotting, Northern blotting, PCR, RT-PCR, qRT-PCR, microarray and sequencing. A kit for in vitro diagnosis of an insulin-related disease, wherein the kit comprises a biomarker comprising at least one microRNA selected from the group consisting of miR- 18a, miR-19b, miR-30d, miR-124, miR-146a, miR-184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof.
A kit for in vitro proliferation of pancreatic β-cells, comprising at least one microRNA selected from the group consisting of miR-18a, miR-19b, miR-30d, miR-124, miR-146a, miR- 184, miR-335, miR-433-5p and miR-503, or a DNA, mRNA or IncRNA thereof.
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