KR102136720B1 - Composition for preventing or treating neurodegenerative disease comprising miR-493-3p inhibitor with inhibitory effect of cell death in hippocampal neural stem cells - Google Patents

Abstract

The present invention relates to a composition for preventing or treating degenerative neurological diseases comprising an miR-141-5p inhibitor or miR-493-3p inhibitor having an inhibitory effect on neural stem cell death. The present inventors performed the microarray-based genome-wide miRNA analysis under conditions according to the presence or absence of insulin using HCN cell-derived total RNAs to confirm the properties of miRNAs. As a result, it was confirmed that when miR-141-5p or miR-493-3p was suppressed, the survival rate of hippocampal-derived neural stem cells could be improved to improve the rate of engraftment of neural stem cells. Therefore, the miR-141-5p inhibitor or miR-493-3p inhibitor of the present invention can be usefully used in the treatment of degenerative cranial nerve disease.

Description

Composition for preventing or treating neurodegenerative disease comprising miR-493-3p inhibitor with inhibitory effect of cell death in hippocampal neural stem cells}

The present invention relates to a composition for preventing or treating degenerative neurological diseases comprising an miR-141-5p inhibitor or a miR-493-3p inhibitor having an inhibitory effect on neural stem cell death, miR-141-5p or miR-493 When -3p is suppressed, the survival of hippocampal-derived neural stem cells can be controlled, thereby improving the rate of engraftment of neural stem cells.

Stem cell-based treatment to regenerate damaged tissue is a new hope for patients with multiple neurological diseases caused by permanent damage to the central nervous system. Accordingly, pluripotent stem cells found in adult brains such as the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus are attracting attention. Adult neural stem cells contribute to functional maturation of adult neurons and maintenance of various brain activities, including normal cognitive function. In addition, it has been reported that the brain-derived hippocampal neural (HCN) stem cells of adult mice maintain the characteristics of pluripotent adult neural stem cells. In addition, HCN cells can differentiate into neurons, astrocytes or oligodendrocytes as well as self-renewal ability. This indicates that adult neural stem cells or HCN cells are highly likely to be used for regenerative treatment of degenerative neurological diseases.

MicroRNAs (miRNAs or miRs) are double-stranded unencoded RNA molecules with about 22-25 nucleotides (nts). It down-regulates gene expression at post-transcriptional levels by inhibiting target mRNA degradation or target mRNA translation. MiRNAs make long primary transcripts of 70-100 nts by RNA polymerase II, which are called pri-miRNAs. Subsequently, mature miRNA duplexes are made by ribonuclease Drosha and Dicer. The miRNA thus generated complementarily binds to the target mRNA and induces translation inhibition and mRNA destabilization, thereby regulating gene expression mainly related to cell proliferation, death, cell metabolism, intracellular signaling, and cell migration. In addition, miRNA expression patterns are tissue-specific and can be defined as the physiological properties of cells in certain genetic or environmental contexts. In addition, abnormal expression of miRNA disrupts normal physiological activity and inevitably leads to a pathological state of cells. Therefore, research on miRNA is gradually focusing on identifying functional characteristics of miRNAs in various human diseases. Nevertheless, the role of miRNA(s) in inducing apoptosis of HCN stem cells has not been established.

United States Patent Publication US2015-0037299 (2015.02.05)

An object of the present invention is to provide a composition for inhibiting neural stem cell death, comprising an miR-141-5p inhibitor or miR-493-3p inhibitor as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative cranial neuropathy, comprising a miR-141-5p inhibitor or miR-493-3p inhibitor as an active ingredient.

Another object of the present invention is to provide a method for screening a therapeutic agent for degenerative cranial nerve disease by measuring the expression level of miR-141-5p or miR-493-3p in neural stem cells.

The present invention provides a composition for inhibiting neural stem cell death, comprising an miR-141-5p inhibitor or miR-493-3p inhibitor as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative cranial neuropathy, comprising miR-141-5p inhibitor or miR-493-3p inhibitor as an active ingredient.

In addition, the present invention comprises the steps of contacting the test substance to the nerve stem cells; Measuring the expression level of miR-141-5p or miR-493-3p in the neural stem cells in contact with the test substance; And it provides a method for screening for a therapeutic agent for degenerative cranial nerve disease, comprising the step of selecting a test substance having a reduced expression level of miR-141-5p or miR-493-3p compared to a control sample.

The present invention relates to a composition for preventing or treating degenerative neurological diseases comprising an miR-141-5p inhibitor or miR-493-3p inhibitor having an inhibitory effect on neural stem cell death. The present inventors performed the microarray-based genome-wide miRNA analysis under conditions according to the presence or absence of insulin using HCN cell-derived total RNAs to confirm the properties of miRNAs. As a result, it was confirmed that when miR-141-5p or miR-493-3p was suppressed, the survival rate of hippocampal-derived neural stem cells could be improved to improve the rate of engraftment of neural stem cells. Therefore, the miR-141-5p inhibitor or miR-493-3p inhibitor of the present invention can be usefully used in the treatment of degenerative cranial nerve disease.

1 is a cell culture results for miRNA array. (A) insulin is present for a specified period of time, (B) insulin is deficient for a specified period of time.
2 shows a sample preparation process for miRNA arrays. (A) Insulin (+) experimental group was prepared with a sample for 24 hours, and in the insulin (-) experimental group, cells cultured in a medium without insulin for 12, 24, and 48 hours over time were prepared with a material, and triple for each experimental group. Samples were prepared (n=3). (B) As a result of analyzing the total RNA sample, agarose gel electrophoresis (1%) is shown. (C) As a result of total RNA sample analysis, the results of Nanodrop analysis using a spectrophotometer [(+) 24H] are shown.
3 shows a list of total RNA samples selected for the miRNA array.
Figure 4 shows Affimetrix Genechip 4.0 version information used in the miRNA array.
Figure 5 shows the results of the miRNA array analysis. (A) As a box plot result, it shows the range of signal intensity obtained from 12 sets of samples used in miRNA array, and it shows the signal intensity of a similar degree in all experimental sets. (B) As a result of Pearson correlation plot, correlation between array chips is analyzed, and when the value is close to 1, it means a positive correlation. (C) This is the result of summarizing the array analysis.
Figure 6 shows the quantitative results of miR-141-5p through RT-qPCR under insulin deficiency conditions (*: 0.05> p, **: 0.005> p).
7 shows the effect of miR-141-5p treatment on apoptosis of rHCN cells. (A) Quantitative apoptosis assay (miRNA concentration: 50 nM), (B) morphological characteristics (48 hour treatment). *: 0.05> p, **: 0.005> p.
8 shows the effect of anti-miR-141-5p treatment on cell death by miR-141-5p. (A) Quantitative apoptosis assay (miRNA concentration: 50 nM, anti-miRNA concentration: 100 nM, 48 hour treatment), (B) morphological characteristics. rno: derived from rat, hsa: derived from human.
9 shows the effect of anti-miR-141-5p treatment on cell death due to insulin deficiency. (A) Quantitative apoptosis assay (anti-miRNA concentration: 100 nM, 48 hour treatment), (B) morphological characteristics. rno: derived from rat, hsa: derived from human.
10 shows the quantitative results of miR-493-3p through RT-qPCR under insulin deficiency conditions (*: 0.05> p, **: 0.005> p).
11 shows the effect of miR-493-3p treatment on apoptosis of rHCN cells. (A) Quantitative apoptosis assay (miRNA concentration: 50 nM), (B) morphological characteristics (48 hour treatment). *: 0.05> p, **: 0.005> p.
12 shows the effect of anti-miR-493-3p treatment on cell death by miR-493-3p. (A) Quantitative apoptosis assay (miRNA concentration: 50 nM, anti-miRNA concentration: 100 nM, 48 hour treatment), (B) morphological characteristics. rno: derived from rat, hsa: derived from human. *: 0.05> p, **: 0.005> p.
13 shows the effect of anti-miR-493-3p treatment on cell death due to insulin deficiency. (A) Quantitative apoptosis assay (anti-miRNA concentration: 100 nM, 48 hour treatment), (B) morphological characteristics. rno: derived from rat, hsa: derived from human. *: 0.05> p, **: 0.005> p.

The present invention provides a composition for inhibiting neural stem cell death, comprising an miR-141-5p inhibitor or miR-493-3p inhibitor as an active ingredient.

Preferably, the miR-141-5p inhibitor or miR-493-3p inhibitor may be any inhibitor capable of inhibiting miRNA, but antagomir, antisense oligonucleotide or RNA inhibitory molecule ( inhibitory RNA molecule), but is not limited thereto. More preferably, the miR-141-5p inhibitor is anti-rno-miR-141-5p represented by SEQ ID NO: 1 (5'-caacacugcacuggaagaugga-3') or SEQ ID NO: 2 (5'-uccaacacuguacuggaagaug-3') May be anti-hsa-miR-141-5p, the miR-493-3p inhibitor may be an anti-rno-miR-493-3p or sequence represented by SEQ ID NO: 3 (5'-cuggcacacaguagaccuuca-3') It may be anti-hsa-miR-493-3p represented by the number 4 (5'-ccuggcacacaguagaccuuca-3').

Preferably, the composition can improve the engraftment rate of neural stem cells.

Preferably, the neural stem cells may be nerve stem cells derived from the hippocampus, but are not limited thereto.

The antagomir or antisense oligonucleotide is characterized by having a sequence complementary to the base sequence of miR-141-5p or miR-493-3p. Inhibitors with sequences complementary to these miRNAs bind to miR-141-5p or miR-493-3p and prevent these miRNAs from functioning.

On the other hand, the miR-141-5p is represented by SEQ ID NO: 5 (5'-uccaucuuccagugcaguguug-3'; miRBase Accession # MIMAT0017128) miR-141-5p derived from rat or SEQ ID NO: 6 (5'-caucuuccaguacaguguugga-3 '; miRBase Accession # MIMAT0004598) may be a human-derived miR-141-5p.

In addition, the miR-493-3p is represented by SEQ ID NO: 7 (5'-ugaaggucuacugugugccag-3'; miRBase Accession # MIMAT0003191) miR-493-3p derived from rat or SEQ ID NO: 8 (5'-ugaaggucuacugugugccagg-3 '; miRBase Accession # MIMAT0003161) may be a human-derived miR-493-3p.

As described above, miR-141-5p or miR-493-3p has a common sequence in human and rat, and since the common sequence is an important part of miRNA targeting, miR- The 141-5p inhibitor or miR-493-3p inhibitor can be applied to humans, and thus can be predicted to be effective in treating degenerative cranial nerve disease.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative cranial neuropathy, comprising miR-141-5p inhibitor or miR-493-3p inhibitor as an active ingredient.

Preferably, the miR-141-5p inhibitor or miR-493-3p inhibitor may be any inhibitor capable of inhibiting miRNA, but antagomir, antisense oligonucleotide or RNA inhibitory molecule ( inhibitory RNA molecule), but is not limited thereto. More preferably, the miR-141-5p inhibitor is anti-rno-miR-141-5p represented by SEQ ID NO: 1 (5'-caacacugcacuggaagaugga-3') or SEQ ID NO: 2 (5'-uccaacacuguacuggaagaug-3') May be anti-hsa-miR-141-5p, and may be anti-rno-miR-493-3p represented by SEQ ID NO: 3 (5'-cuggcacacaguagaccuuca-3') or SEQ ID NO: 4 (5'-ccuggcacacaguagaccuuca- 3') may be anti-hsa-miR-493-3p.

Preferably, the degenerative cerebral neuropathy is dementia, Alzheimer's disease, stroke, Parkinson's disease, Huntington's disease, Pick's disease, Amyotrophic lateral sclerosis (ALS). , Creutzfeldt-Jakob disease (CJD), Progressive supranuclear palsy, Multiple system strophy, Olive-pontocerebellar atrophy (OPCA), Shire-Dragger Syndrome (Shy-Drager syndrome), Essential tremor, Cortico-basal ganlionic degeneration, Diffuse Lewy body disease, and striatonigral degeneration However, it is not limited thereto.

The pharmaceutical composition of the present invention may include chemicals, nucleotides, antisense, siRNA oligonucleotides, and natural product extracts as active ingredients. The pharmaceutical composition or combination preparation of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanders, lubricants , Solubilizing agents such as lubricants or flavoring agents. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include, as sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

Pharmaceutical formulation forms of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of active compounds, etc. Can. The pharmaceutical composition of the present invention is a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of diseases. Thus, the type of disease, the severity of the disease, the type and content of active and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health status, gender and diet, time of administration, route of administration and composition It can be adjusted according to various factors, including secretion rate, duration of treatment, and drugs used simultaneously. Without being limited thereto, for example, in the case of an adult, when administered once to several times a day, the inhibitor of the present invention is administered once to several times a day, when the compound is 0.1ng/kg to 10g/kg, a polypeptide, In the case of protein or antibody, 0.1ng/kg~10g/kg, antisense nucleotide, siRNA, shRNAi, miRNA can be administered at a dose of 0.01ng/kg~10g/kg.

In addition, the present invention comprises the steps of contacting the test substance to the nerve stem cells; Measuring the expression level of miR-141-5p or miR-493-3p in the neural stem cells in contact with the test substance; And it provides a method for screening for a therapeutic agent for degenerative cranial nerve disease, comprising the step of selecting a test substance having a reduced expression level of miR-141-5p or miR-493-3p compared to a control sample.

The term "test substance" used while referring to the screening method of the present invention means an unknown candidate substance used in screening to examine whether it affects the expression level of a gene or the expression or activity of a protein. do. The test substance includes, but is not limited to, chemicals, nucleotides, antisense-RNA, miRNA, small interference RNA (siRNA), and natural product extracts.

Hereinafter, the present invention will be described in detail according to examples not limiting the present invention. It should be understood that the following examples of the present invention are only intended to embody the present invention and do not limit or limit the scope of the present invention. Therefore, from the detailed description and examples of the present invention, what can be easily inferred by experts in the technical field to which the present invention pertains is interpreted as belonging to the scope of the present invention.

< Experimental Example >

The following experimental examples are intended to provide an experimental example commonly applied to each embodiment according to the present invention.

One. HCN Cell culture

Adult mouse brain-derived hippocampal neural (HCN) stem cells were isolated as previously reported (STEM CELLS 2008;26:2602-2610). All culture plates were poly-L-ornithine (10 μg/ml plastic plate and 50 μg/ml glass slide; Sigma-Aldrich, St. Louis, MO) and mouse laminin (5 μg/ml; BD Pharmingen, San Diego, CA ). Ham's F-12 medium (Invitrogen), 1 mM L-glutamine (Invitrogen), 100 μg/ml streptomycin, 100 U/ml penicillin (Invitrogen), 20 ng/ml b-FGF (PeproTech, Rocky Hill, NJ) and 5 Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with self-made N2 supplement containing mg/L insulin (Sigma-Aldrich), 16 mg/L putrescin dihydrochloride (Sigma-Aldrich), 100 mg/L transferrin HCN cells were cultured in serum-free medium containing (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich) and 20 nM progesterone (Sigma-Aldrich). The medium was adjusted to pH 7.2 using sodium bicarbonate (1.27 g/L, Sigma-Aldrich). Insulin-free insulin-free medium was prepared. Cells were passaged at a ratio of 1:4-6. Cells were cultured at 37° C. under 5% CO ₂ conditions. Insulin-containing medium was indicated by I (+), and the medium without insulin was indicated by I (-).

2. miRNAs And cell infection

miRNA mimics are double helix RNA molecules that have the same sequence and function as the intrinsic miR-141-5p or miR-493-3p. To inhibit related miR mimics, Anti-miRs chemically synthesized double-stranded nucleic acids and introduced into cells through transfection. Negative controls (miR-Con and anti-miR-Con) were purchased from Genolution (Seoul, Korea) and Dharmacon (Lafayette, CO). For transient transfection, HCN cells were seeded and miRNAs were transfected using Lipofectamine 2000 (Invitrogen).

3. Total RNA extraction

Total RNA was extracted from HCN cells cultured for 12, 24, 48 hours depending on the presence or absence of insulin (FIGS. 1 and 2). In an RNA-free microcentrifuge tube, approximately 2×10 ⁶ cells were treated with 0.5 ml Trizol reagent (Invitrogen). After centrifugation at 12,000 g for 15 minutes at 4° C., the supernatant was transferred to a new tube containing 0.2 ml chloroform. The tube was shaken strongly and reacted at room temperature for 5 minutes. After centrifugation, the supernatant was transferred to a new tube containing pure isopropyl alcohol in the same amount and reacted at room temperature for 30 minutes. After centrifugation, the supernatant was discarded and 1.0 ml of 70% ethanol was added to the RNA pellet for washing. The ethanol was removed by centrifugation, and the residual RNA pellet was suspended in DEPC-treated RNase-free water. The total RNA amount and quality obtained were measured by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and agarose gel electrophoresis, respectively (FIG. 2).

4. miRNA Microarray And data analysis

Total RNA samples for miRNA microarray analysis were sent to DNALink (Seoul, Korea) (Figure 3). The quality and concentration of total RNA samples were confirmed by Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) and NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific), respectively. The RIN value of total RNA, indicating the degree of RNA integrity, was 9.8 to 10.0, and the minimum value >8.0 required for microarray analysis was sufficiently met. In addition, even in the initial electropherogram profile, the RNA sample was found to contain sufficient low molecular weight RNA. Thereafter, microarray was performed using Affymetrix GeneChip miRNA 4.0 Arrays (Santa Clara, CA). FlashTag Labeling Kit was used for probe-labeling and Affymetrix Expression Console Software was used for data analysis (FIG. 4). . The total number of mature miRNA probes per array was 30,324 including 728 rat mature miRNAs. After background removal, normalization and detection evaluation, a signal was shown (FIG. 5 ). Statistical significance was P-value <0.05 and fold-change cutoff was >1.3.

5. miRNA About real time Reverse warrior Quantitative PCR (RT- qPCR ) analysis

Quantitative analysis of selected mature miRNA expression levels was performed via RT-qPCR. RT was performed using a mir-X miRNA first-strand synthesis kit (Clontech, Mountain View, CA). RT-qPCR to quantify miRNA expression was performed using IQ SyBr Green I mix (Bio-Rad, Hercules, CA). Forward 5'specific PCR primers and 3'mRQ primers complementary to miRNAs were used. The 5'primer was prepared in the same manner as the corresponding miRNA sequence, and the 3'mRQ primer was used as the mRQ primer provided by the RT-qPCR kit. RT-qPCR conditions are as follows. RT was performed at 37°C for 1 hour, and as a template, qPCR was performed at 95°C, 10 seconds (initial denaturation), and 40 cycles of 95°C, 5 seconds and 60°C, and 20 seconds. U6 RNA was used as an intrinsic control.

6. Hoechst 33342/ propidium Cell death is measured after iodide (PI) staining

The cell viability/killing of HCN cells was morphologically observed under a light microscope, and the degree of cell death was measured by Hoechst 33342/PI staining (Thermo Fisher Scientific). At the specified time, 5 μg/ml Hoechst 33342 and PI solution were added to the culture medium, and the cells were kept at 37° C. for 1 hour under 5% CO ₂ conditions. After 5 fields were randomly photographed under a fluorescence microscope, the cell death rate was measured by calculating the ratio of PI-positive and Hoechst 33342-positive cells.

Cell death (%) = [PI-positive cell count (red) / Hoechst33342-positive cell count (blue)] × 100

< Example 1> HCN Stem cell culture microRNA Effect of insulin deficiency on expression patterns

HCN cells within 1 day after insulin deficiency, cell proliferation delay, cell shrinkage (cell shrinkage), cell process damage, cell death occurs. The degree of cell death was most severe at 2-3 days, and the cells eventually separated from the culture plate (FIG. 1 ). In order to confirm whether a specific characteristic appears in miRNA expression during the cell death process according to insulin deficiency, miRNA microarray analysis was performed (FIGS. 2 and 4 ). In order to minimize the experimental deviation, three independent tests were performed per culture condition with or without insulin. The signal intensity variation for each array was significantly reduced after quantile normalization as shown in the box plot (Figure 5A). The correlation coefficient between the microarray data sets was 0.97 or more, which shows excellent reproducibility, as shown in the correlation scatter plot (FIG. 5B). Total miRNA levels were filtered based on p-value cutoff 0.05. As a result of testing 728 rat mature miRNAs, in the absence of insulin compared to the case where insulin was added, a total of 175 miRNAs showed significant differences in expression levels. After insulin deficiency, 30 miRNAs were up-regulated and 145 miRNAs were down-regulated (fold-change> 1.3, Figure 5C). Summarizing the results, it shows that after insulin deficiency, a difference occurs in the miRNA expression profile during the HCN cell death process.

As a result of the miRNA array, the increase group increased by more than 1.5 was 6-21 species per hour, and the targets decreased more than 0.66 were identified as 57-109 species per hour. Among them, additional experiments were performed by selecting miR-141-5p and miR-493-3p. In addition, the degree of expression increase was confirmed through RT-qPCR (*: 0.05> p, **: 0.005> p).

Detailed information on miR-141-5p or miR-493-3p used in the present invention is shown in the table below.

< Example 2> Under insulin deficiency conditions, RT- qPCR through miR Quantification of -141-5p

RT-qPCR confirmed the increase phenomenon confirmed in the array. rHCN cells were cultured for 24 or 48 hours depending on the presence or absence of insulin, and RT-qPCR was specifically performed for the indicated miRNAs (n≧3). The expression level of miR-141-5p was improved in insulin deficiency condition (Fig. 6). Each value is expressed as mean±SD. U6 RNA was used as an intrinsic control.

< Example 3> HCN cell Viability Stay up-regulated miR Effect of -141-5p treatment

On HCN cells, cells were transfected and observed with synthetic miRNA mimics in the presence of insulin to characterize the physiological role of the up-regulated miR-141-5p. Unlike the control group to which the control miRNA mimic was added, the cells significantly lost viability in the presence of the up-regulated miRNA mimics, which was confirmed through a marked increase in PI-positive cells.

The degree of cell death by miR-141-5p was similar to that of insulin deficiency (Fig. 7). These results indicate that in the absence of insulin, up-regulated miR-141-5p may be involved in the regulation of HCN cell viability.

< Example 4> miR Anti-miR-141-5p that inhibits -141-5p-induced cell death and protects cell death induced by insulin deficiency

To verify the specificity of miR-141-5p in HCN cell death, we used anti-miR-141-5p with a complementary sequence to miR-141-5p as a miR-141-5p inhibitor, which The physiological activity of miR-141-5p is knocked down (FIG. 8 ). When anti-miR-141-5p (rno-141-5p inhibitor/hsa-141-5p inhibitor) and miR-141-5p were treated simultaneously, cell death was significantly reduced. Anti-miR-Con with a non-specific mixed sequence did not affect cell death induced by miR-141-5p mimic.

On the other hand, it was confirmed that anti-miR-141-5p (rno-141-5p inhibitor/hsa-141-5p inhibitor) reduces the apoptosis-inducing effect under insulin deficiency (FIG. 9).

< Example 5> Under insulin deficiency conditions, RT- qPCR through miR Quantification of -493-3p

RT-qPCR confirmed the increase phenomenon confirmed in the array. rHCN cells were cultured for 48 hours depending on the presence or absence of insulin, and RT-qPCR was specifically performed for the indicated miRNAs (n≧3). The expression level of miR-493-3p was improved in insulin deficiency condition (Fig. 10). Each value is expressed as mean±SD. U6 RNA was used as an intrinsic control.

< Example 6> HCN cell Viability Stay up-regulated miR Effects of -493-3p processing

On HCN cells, cells were transfected and observed with synthetic miRNA mimics in the presence of insulin to characterize the physiological role of up-regulated miR-493-3p. Unlike the control group to which the control miRNA mimic was added, the cells significantly lost viability in the presence of the up-regulated miRNA mimics, which was confirmed through a marked increase in PI-positive cells.

The degree of cell death by miR-493-3p was similar to that of insulin deficiency (Fig. 11). The results indicate that in the absence of insulin, up-regulated miR-493-3p may be involved in the regulation of HCN cell viability.

< Example 7> miR Anti-miR-493-3p inhibits -493-3p-induced cell death and protects cell death induced by insulin deficiency

To verify the specificity of miR-493-3p in HCN cell death, we used anti-miR-493-3p with a complementary sequence to miR-493-3p as a miR-493-3p inhibitor. The physiological activity of miR-493-3p is knocked down (FIG. 12 ). When anti-miR-493-3p (rno-493-3p inhibitor/hsa-493-3p inhibitor) and miR-493-3p were treated simultaneously, cell death was significantly reduced. Anti-miR-Con with non-specific mixed sequences did not affect cell death induced by miR-493-3p mimic.

On the other hand, it was confirmed that anti-miR-493-3p (rno-493-3p inhibitor/hsa-493-3p inhibitor) reduces the apoptosis-inducing effect under insulin deficiency (FIG. 13).

<110> University of Ulsan Foundation For Industry Cooperation THE ASAN FOUNDATION <120> Composition for preventing or treating neurodegenerative disease comprising miR-493-3p inhibitor with inhibitory effect of cell death in hippocampal neural stem cells <130> ADP-2018-0442 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 22 <212> RNA <213> rat <400> 1 caacacugca cuggaagaug ga 22 <210> 2 <211> 22 <212> RNA <213> human <400> 2 uccaacacug uacuggaaga ug 22 <210> 3 <211> 21 <212> RNA <213> rat <400> 3 cuggcacaca guagaccuuc a 21 <210> 4 <211> 22 <212> RNA <213> human <400> 4 ccuggcacac aguagaccuu ca 22 <210> 5 <211> 22 <212> RNA <213> rat <400> 5 uccaucuucc agugcagugu ug 22 <210> 6 <211> 22 <212> RNA <213> human <400> 6 caucuuccag uacaguguug ga 22 <210> 7 <211> 21 <212> RNA <213> rat <400> 7 ugaaggucua cugugugcca g 21 <210> 8 <211> 22 <212> RNA <213> human <400> 8 ugaaggucua cugugugcca gg 22

Claims (6)

In vitro containing the miR-493-3p inhibitor, anti-rno-miR-493-3p represented by SEQ ID NO: 3 or anti-hsa-miR-493-3p represented by SEQ ID NO: 4 as an active ingredient (in vitro ) Reagent composition for inhibiting neural stem cell death. The reagent composition for inhibiting neural stem cell death in vitro, according to claim 1, wherein the composition improves the engraftment rate of the neural stem cells. The reagent composition for inhibiting neural stem cell death in vitro according to claim 1 or 2, wherein the neural stem cell is a hippocampal-derived neural stem cell. delete delete delete

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