WO2018210259A1 - Medication for regulating appetite and body weight, and application thereof - Google Patents

Abstract

Disclosed in the present invention is an application of an inhibitor capable of inhibiting the function of miR-183 in manufacture of medications used for increasing body weight, improving gastrointestinal function, and increasing subcutaneous fat and the content of fat in skin cells. Also disclosed are an inhibitor capable of inhibiting the function of miR-183, and a pharmaceutical composition and kit comprising the inhibitor.

Description

[Name of invention established by ISA according to Rule 37.2] A drug regulating appetite and body weight and their application Technical field

The present invention relates to the field of biomedicine, and in particular to an application for inhibiting the function of miR-183 in increasing body weight, enhancing appetite and gastrointestinal function, increasing fat content in skin and skin cells, and inhibiting the function of miR-183. Method, a miR-183-based inhibitor, pharmaceutical composition and kit, and their use in inhibiting the function of miR-183, particularly in the prevention and/or treatment of wasting, anorexia, indigestion, weak gastrointestinal function The use of at least one of skin care, skin elasticity and gloss.

Background technique

Previous studies have shown that the brain (the hypothalamus is particularly important) - the central axis of the gastrointestinal tract plays an important regulatory role in appetite, digestion and absorption, and glucose and lipid metabolism ^[1] , so the gene expression in the hypothalamus-gastrointestinal is very variable with ageing. May be the main reason for the elderly to lose weight, weak gastrointestinal function, low subcutaneous fat content and dry skin wrinkles. In addition, studies have shown that the brain-gastrointestinal-hepatic central axis plays an important regulatory role in glycolipid metabolism ^[2] , indicating that the brain-gastrointestinal-hepatic central axis may be a new target organ system for metabolic diseases ^[3] .

There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of energy metabolism ^[4] . MicroRNAs are a class of non-coding RNA molecules (www.mirbase.org) that are 16-25 nt in length and are capable of recognizing and silencing RNA expression and/or protein expression of a target gene by complementary pairing with a target gene. After the mature microRNA is loaded onto the RNA-induced silencing complex (RISC), it is combined with the complementary sequence in the 3'-UTR of the target gene mRNA by base pairing, thereby triggering the degradation of the mRNA and/or inhibiting its protein. translation. The nucleotides from the second to the eighth position of the 5' end of the microRNA are called "core sequences". The complementary pairing of these seven nucleotides with the target gene is the key to recognition of the target gene. The higher the degree of pairing, the binding and The greater the likelihood and ability to regulate a target gene. Simultaneous pairing of sequences other than the "core sequence" of the microRNA with the target gene also enhances its ability to bind and regulate the target gene. It is precisely because microRNAs recognize and regulate the expression of target genes through incomplete pairing that a microRNA can simultaneously regulate multiple target genes to different degrees in one cell.

Summary of the invention

The object of the present invention is to develop microRNA-based drugs for controlling appetite, digestion and absorption, and glycolipid metabolism.

In order to achieve the above object, in one aspect, the present invention provides an application for inhibiting the function of miR-183 in increasing body weight, enhancing appetite and gastrointestinal function, increasing fat content in skin fat and skin cells, particularly in preparation for prevention and/or Or for the treatment of drugs and/or foods that are thin, anorexia, indigestion, weak in gastrointestinal function, or in skin care products for skin care, skin elasticity and luster.

In a second aspect, the invention provides a method of inhibiting the function of miR-183, wherein the method comprises: contacting a miR-183 inhibitor with a target cell expressing miR-183.

In a third aspect, the present invention provides a miR-183 inhibitor, wherein the miR-183 inhibitor is an antisense oligonucleotide comprising antisense DNA and antisense RNA, the antisense oligonucleotide Complementary to miR-183 and having a length of 8-40 nucleotides; or the miR-183 inhibitor is a small interfering RNA of the miR-183 precursor.

In a fourth aspect, the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a miR-183 inhibitor as described above and a pharmaceutically acceptable carrier.

In a fifth aspect, the present invention also provides a kit, wherein the kit comprises a miR-183 inhibitor as described above, optionally, the kit further comprising a pharmaceutically acceptable carrier.

In a sixth aspect, the present invention provides a method as described above, a miR-183 inhibitor as described above, a pharmaceutical composition as described above and/or a kit as described above, in inhibiting the function of miR-183 Application; especially in the application of increasing body weight, enhancing appetite and gastrointestinal function, increasing subcutaneous fat and fat content in skin cells; more preferably in preventing and/or treating wasting, anorexia, indigestion, weak gastrointestinal function, skin maintenance, enhancement Application in at least one of skin elasticity and gloss.

In a seventh aspect, the present invention provides the use of a miR-183 inhibitor as described above, a pharmaceutical composition as described above, for the preparation of a medicament for inhibiting the function of miR-183; in particular, for preparation for prevention and / or treatment of weight loss, anorexia, indigestion, weak gastrointestinal drugs and / or food, or for the application of skin care, skin elasticity and luster.

The present invention can fully inhibit the function of miR-183 by contacting a miR-183 inhibitor with a target cell expressing miR-183 (including inhibiting the binding of miR-183 to its target gene or reducing the expression level of miR-183, thereby inhibiting The function of miR-183), when used for individual administration, can effectively prevent and/or treat diseases caused by an increase in the expression level of miR-183, for example, wasting, anorexia, indigestion, weak gastrointestinal function, subcutaneous fat Less diseases. The therapy provided by the invention can comprehensively regulate appetite, gastrointestinal digestion and absorption function and glycolipid metabolism, and is an oligonucleotide; thus, the effect is strong and the side effects are small.

Other features and advantages of the invention will be described in detail in the detailed description which follows.

DRAWINGS

The drawings are intended to provide a further understanding of the invention, and are intended to be a part of the description of the invention. In the drawing:

Figure 1 is a graph showing the expression levels of miR-183 in the hypothalamus, stomach and liver, wherein the expression level of miR-183 in 12-month-old mice is significantly higher than that in 2-month-old mice. .

Figure 2A is a linear plot of miR-183 ASO dose versus its inhibitory effect on miR-183.

Figure 2B is a comparison of the functional effects of miR-183 ASO, random control nucleotides, miR-183 mismatched ASO at the cellular level to inhibit miR-183.

Figure 3A is a graph comparing the changes in body weight over time in mice administered with miR-183 ASO and PBS, respectively.

Figure 3B is a graph comparing the rate of weight gain over time in mice administered with miR-183 ASO and PBS, respectively.

Figure 4 is a graph comparing the food intake of mice administered with miR-183 ASO and PBS, respectively.

Figure 5 is a graph comparing the weight of kidney, epididymal fat and stomach of mice fed miR-183 ASO and PBS, respectively, for three months.

Figure 6 is a graph comparing the effect of miR-183 siRNA and random control RNA on miR-183 expression.

detailed description

Specific embodiments of the present invention will be described in detail below. It is to be understood that the specific embodiments described herein are merely illustrative and not restrictive.

Unless otherwise stated, the technical terms used herein have the same meaning as the terms commonly understood by those skilled in the art.

The inventors of the present invention found in the course of the study that miR-183 in the hypothalamic-gastric axis of the dominant appetite, digestion and absorption and energy metabolism is in the 12-month-old mouse compared to the 2 month old mouse. Significantly upregulated, and expression in the liver is also significantly upregulated. Based on the above findings, the inventors used the TargetScanHuman (www.targetscan.org) algorithm to predict the target gene of miR-183 that is conserved in vertebrates, and then compared with the KEGG pathway, many target genes of miR-183 are enriched in gastric acid. Secretion, salivation, phosphatidylinositol signaling pathway, insulin signaling pathway and type 2 diabetes signaling pathway (Table 1). Another 68 miR-183 target genes are located in the MAPK signaling pathway involved in adipocyte differentiation, cholesterol transport, and type 2 diabetes ^[5,6] ; 84 miR-183 targets are involved in the regulation of glucose metabolism, fat The cell-differentiated PI3K-Akt signaling pathway ^[5,7] ; and 26 miR-183 targets are located in the TGF-beta signaling pathway associated with obesity, diabetes, and liver disease ^[5,8,9] ; and 34 miR- The target of 183 is located in the GnRH signaling pathway that regulates blood glucose and metabolism [10] (Table 1).

Table 1

Based on the above studies, the inventors of the present invention found that miR-183 can serve as a marker of appetite and gastrointestinal function, and subsequently demonstrated that appetite can be enhanced and the function, body weight and fat weight of the stomach can be increased by inhibiting the function of miR-183.

Accordingly, the present invention provides the use of the inhibitory marker miR-183 for preventing and/or treating the following diseases and/or symptoms: weight loss, anorexia, dyspepsia, weak gastrointestinal function, less subcutaneous fat, and reduction of subcutaneous fat. Skin wrinkles and dry skin, maintain skin, increase skin elasticity and shine. The present invention also provides a miR-183 inhibitor, a method for inhibiting the function of miR-183 by the miR-183 inhibitor, and a pharmaceutical composition and kit comprising the miR-183 inhibitor, and they are increasing Application in body weight, appetite and gastrointestinal function, increase in subcutaneous fat and fat content in skin cells; more preferably in prevention and/or treatment of wasting, anorexia, indigestion, weak gastrointestinal function, skin maintenance, skin elasticity and luster Application in at least one; further preferably in the preparation of a medicine and/or food for preventing and/or treating wasting, anorexia, indigestion, weak gastrointestinal function, or a skin care product for maintaining skin, enhancing skin elasticity and luster Application in .

In the present invention, the term "weak gastrointestinal function" refers to a decline in the gastrointestinal function of an individual compared to gastrointestinal function in a normal or daily state, for example, including a diminished function of digestion and absorption of the gastrointestinal system.

method

The present invention provides a method of inhibiting the function of miR-183, wherein the method comprises: contacting a miR-183 inhibitor with a target cell expressing miR-183.

In a preferred aspect, miR-183 has the nucleotide sequence set forth in SEQ ID No: 1 (human and murine miR-183 sequences are identical) (UAUGGCACUGGUAGAAUUCACU).

According to the present invention, the "inhibition of the function of miR-183" refers to the degree of down-regulation of miR-183 expression of its target gene in a target cell expressing miR-183 of the same species which is not treated by the method of the present invention, The miR-183-expressing target cells treated by the present invention have a reduced degree of down-regulation of their target gene expression by at least 0.5-fold, and can generally be reduced by at least a factor of two, such as Figures 2A and 2B.

The present invention provides a method of inhibiting miR-183 function in a target cell expressing miR-183 in vivo or in vitro. The term "inhibiting the function of miR-183" means that the expression level of a target gene regulated by miR-183 is increased by directly or indirectly acting on miR-183 with an agent. Methods include, but are not limited to, the following:

1) Small molecule compounds

MiR-183 inhibitors include, but are not limited to, naturally occurring or synthetic small molecule compounds that act directly on miR-183 to increase the expression of a target gene regulated by miR-183, usually molecular weight. An organic compound greater than 50 and less than 2500 Daltons. Such candidate compounds possess functional groups that interact with proteins, particularly hydrogen bonds, and typically comprise at least one amine, carbonyl, hydroxyl or carboxyl group. These small molecule miR-183 inhibitors can be found by suitable screening methods or other methods.

2) Antisense oligonucleotide

The antisense oligonucleotide is capable of inhibiting the function of the target miR-183 by direct binding to the target miR-183, including antisense RNA and antisense DNA. Preferably, the antisense oligonucleotide is complementary to miR-183, has a length of 8-40 nucleotides, and has a sequence complementary to nucleotides 2-8 of miR-183.

As described in the Background section, it is well known that microRNAs can recognize and silence the expression and/or translation of a target gene by complementary pairing with a target gene. Similarly, miR-183 can also bind to a partially complementary core. The nucleotide sequence competitively inhibits its own function, thereby upregulating the expression of the target gene of miR-183. Therefore, in the present invention, the term "complementary" includes not only complete complementarity but also partial complementarity as long as it can bind to miR-183 and inhibit its function.

Thus, the antisense oligonucleotide has the following nucleotide sequence:

a) the nucleotide sequence shown in SEQ ID No: 4 (AGTGAATTCTACCAGTGCCATA);

b) A nucleotide sequence which is deleted, substituted or added with one or several nucleotides and which is complementary to miR-183 in the nucleotide sequence shown by SEQ ID No: 4.

When in the case of incomplete complementation, that is, when the antisense oligonucleotide is deleted, substituted or added by one or several nucleotides in the nucleotide sequence shown in SEQ ID No: In the case of the obtained nucleotide sequence, the antisense oligonucleotide preferably has at least 60%, 65%, 70%, 75%, 80%, 85 with respect to miR-183 in the region of the complementary nucleotide. %, 90% or 95% complement each other. More preferably, in the nucleotide region of 2-8 of miR-183, the antisense oligonucleotide has at most 3 nucleotides mismatched with miR-183.

As described above, in the case where the antisense oligonucleotide is not completely complementary to miR-183, it is further preferred that there are at most 10, 9 in length compared to SEQ ID No: 4. A difference of 8, 7, 6, 5, 3, 2 or 1 nucleotides.

In a preferred aspect, the antisense oligonucleotide that is not fully complementary to miR-183 has the nucleotide sequence set forth in SEQ ID No: 5 (AGTGAGCTCTACCAGTGGCATA).

In addition, the present invention also encompasses some conventional modifications of the antisense oligonucleotide to improve the stability and activity of the antisense oligonucleotide, all of which are within the scope of the invention.

It is to be noted in the present invention that fully or incompletely complementary RNA having the above characteristics is also within the scope of the present invention. In view of the stability in cells, it is preferred in the present invention that the antisense oligonucleotide is DNA.

Since the antisense oligonucleotide is capable of being complementary (fully complementary or partially complementary) to miR-183, when the antisense oligonucleotide is contacted with a target cell expressing miR-183 in vivo or in vitro, The antisense oligonucleotide is capable of complementary pairing with miR-183 and inhibits the binding of miR-183 to its target gene (ie, inhibits the activity of miR-183), thereby breaking the silencing of miR-183 on its target gene. .

According to the invention, the method comprises introducing an effective amount of an antisense oligonucleotide complementary to miR-183 into a target cell expressing miR-183. Wherein, the "effective amount" differs depending on the target cell expressing miR-183, and exhibits a certain dose effect, as shown in FIG. 2A of the present invention, according to conventional experimental means by those skilled in the art. And the intended purpose achieved can readily determine the effective dose for target cells expressing miR-183.

When the contact is in vivo contact, the antisense oligonucleotide of the present invention can be administered to an individual by conventional nucleic acid administration. For example, administration of the antisense oligonucleotide can be carried out using the following method: the antisense oligonucleotide can be administered orally or by viral infection, microinjection, or vesicle fusion, or Muscle administration of the antisense oligonucleotide can also be carried out by jet injection. Alternatively, the antisense oligonucleotide may be applied to gold particles and then transdermally administered by a known method such as a particle bombardment apparatus or a "gene gun". These are all technical means conventional in the art, and the present invention will not be repeated here.

Furthermore, the antisense oligonucleotide can also be introduced into a target cell expressing miR-183 by a method of expressing a vector. Such expression vectors have a restriction site located adjacent to the promoter sequence to facilitate insertion of the antisense oligonucleotide. Wherein, the transcription cassette located in the expression vector may include a transcription initiation region, a target gene or a fragment thereof, and a transcription termination region. The vector can be, for example, but not limited to, a plasmid, a virus, etc., and can be selected by a person skilled in the art according to actual conditions.

Furthermore, the antisense oligonucleotides can also be introduced into target cells expressing miR-183 by means of respiratory spray administration, for example by preparation into a spray formulation.

In addition, the antisense oligonucleotide may also be introduced into a target cell expressing miR-183 by oral administration, for example, by preparation into an oral preparation, or by the antisense The oligonucleotide is administered orally in a manner that is mixed with the food.

An individual as described above may be any mammalian cell, including but not limited to: ungulates, eg, cows, goats, pigs, sheep, etc.; rodents, eg, hamsters, mice, rats, rabbits; primates For example, monkeys, baboons, humans, etc.

When the contact is in vitro contact, the antisense oligonucleotide or a vector containing the antisense oligonucleotide (for example, a drug containing the antisense oligonucleotide) can be directly added to The medium in which the target cell expressing miR-183 is cultured is contacted, and the target cell expressing miR-183 into which the antisense oligonucleotide is introduced is cultured under conventional cell culture conditions.

3) RNAi reagent

In a representative embodiment, the RNAi agent targets a precursor molecule of miR-183 (pre-microRNA (human sequence), as shown in SEQ ID No: 2, CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCACUGGUAGAAUUCACUGUGAACAGUCUCAGUCAGUGAAUUACCGAAGGGCCAUAAACAGAGCAGAGACAGAUCCACGA), through the mechanism of RNA interference To regulate the expression of miR-183, that is, indirectly inhibit the function of miR-183.

It is well known in the art that RNA interference (RNAi) is a phenomenon in which homologous mRNA is efficiently and specifically degraded by double-stranded RNA (dsRNA). Since RNAi technology can specifically knock out or turn off the expression of specific genes, this technology has been widely used to explore the field of gene function and treatment of infectious diseases and malignant tumors. In particular, the present application, by using interfering RNA of the precursor molecule of miR-183, causes gene silencing of the precursor molecule of miR-183, thereby reducing the level of the precursor molecule of miR-183, thereby reducing The level of mature miR-183 transformed from the precursor molecule of miR-183, that is, inhibits the function of miR-183, thereby increasing the expression level of the miR-183 target gene.

The RNAi agent can be a small RNA molecule, usually a single-stranded deoxyoligonucleotide (shRNA) that theoretically forms a small hairpin structure, typically no more than 100 nucleotides in length, typically No more than 75 nucleotides; or a 15-30 bp double-stranded deoxyoligonucleotide (siRNA), most typically 20-23 bp, as described in Example 5 of the present invention (eg SEQ) The antisense strand represented by ID No: 7 and the sense strand as shown in SEQ ID No: 8.

In some applications, the RNAi agent can also be a template DNA encoding shRNA or siRNA. These template DNA may be present in a vector, such as a plasmid vector or a viral vector; or may be absent from the vector, but a template DNA encoding shRNA or siRNA plus a common promoter sequence fragment that controls its transcription.

Wherein, the contact of the RNAi agent with the target cell expressing miR-183 may also be in vivo contact or in vitro contact. The method of administering the RNAi agent can be carried out with reference to the description of the antisense oligonucleotide as described above, and the present invention will not be described in detail herein in order to avoid unnecessary repetition.

miR-183 inhibitor

The present invention also provides a miR-183 inhibitor, the specific type of the miR-183 inhibitor may be at least one of a small molecule compound, an antisense oligonucleotide and an RNAi reagent as described above, in order to avoid unnecessary Repeated, the present invention will not be described in detail herein.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising the miR-183 inhibitor as described above and a pharmaceutically acceptable carrier.

In the composition of the present invention, the content of the miR-183 inhibitor as described above as an active ingredient may vary within a wide range, and may be, for example, 0.01 to 99% by weight, preferably, may be 1-70. The weight %, more preferably, may be 5 to 30% by weight.

According to the present invention, the pharmaceutical composition can be prepared into various dosage forms conventional in the art, and the present invention is not particularly limited thereto, and for example, it can be formulated into a solid, semi-solid, liquid or gaseous form, for example, a tablet. , capsules, elixirs, suspensions, syrups, powders, granules, ointments, suppositories, injections, inhalants, aerosols, and the like, which are not enumerated herein.

Therefore, various forms of administration may be carried out depending on the pharmaceutical dosage form, such as, but not limited to, oral administration, buccal administration, rectal administration, parenteral administration, intraperitoneal administration, and respiratory administration. , intradermal administration, transdermal administration.

Wherein, the pharmaceutically acceptable carrier can be selected differently depending on the dosage form, which are well known to those skilled in the art. For example, without limitation, the pharmaceutically acceptable carrier can be starch, gum, lactose, glucose, sucrose, microcrystalline cellulose, kaolin, mannitol, dibasic calcium phosphate, sodium chloride, alginic acid, and the like.

In addition, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives may also be added.

Additionally, the pharmaceutically acceptable carrier may further comprise a targeting agent capable of enhancing targeting of the antisense oligonucleotide to a particular organ or tissue or cell, such as a targeting peptide, and may also Included are transmembrane agents that are capable of carrying the antisense oligonucleotides more readily into target cells expressing miR-183, such as transmembrane peptides, liposomes, microvesicles, and membrane lipoproteins.

According to the present invention, a flavoring agent such as peppermint, wintergreen oil or the like may be added to the pharmaceutical composition. In addition, coloring agents may also be added to the pharmaceutical composition to impart a certain degree of attractiveness to the prepared dosage form or to distinguish it from other products.

According to the present invention, the antisense oligonucleotide can also be combined with other conventional drugs capable of similar effects to prepare a combination pharmaceutical composition.

Kit

The invention provides a kit, wherein the kit comprises an antisense oligonucleotide as described above, optionally, the kit further comprises an additional reagent, for example, pharmaceutically acceptable as described above Accepted carriers, flavoring and/or coloring agents, solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizers, preservatives, targeting agents or transmembrane agents.

According to the present invention, the additional reagent may be present in the kit in combination with the antisense oligonucleotide, or may be stored separately in the kit and mixed again when used.

The kit of the present invention may further include an instruction manual, and the form of the specification is not particularly limited, and may be, for example, a printed paper form, a CD form, or a web address. Get access to it via the internet.

application

The present invention provides for inhibiting the function of miR-183 (an appetite and gastrointestinal function marker) for preventing and/or treating at least one of the following diseases and/or symptoms, and/or for maintaining skin, enhancing skin elasticity and gloss Applications.

The diseases and/or symptoms include: wasting, anorexia, indigestion, weak gastrointestinal function or less subcutaneous fat, and symptoms similar to those of these diseases.

In particular, the use includes the preparation of a medicament and/or food for the prevention and/or treatment of any of the above diseases and/or symptoms, or a cosmetic skin care product. Wherein the food product comprises a health care product.

The invention also provides the use of a miR-183 inhibitor as described above, a pharmaceutical composition as described above, a kit as above and/or a method as described above for inhibiting the function of miR-183.

Further preferably, the use comprises preventing and/or treating any of the above diseases and/or symptoms.

Further, the present invention provides the use of the miR-183 inhibitor as described above, the pharmaceutical composition as described above, in the preparation of a medicament for reducing the amount of miR-183.

Preferably, the medicament comprises a medicament and/or a food for preventing and/or treating any of the above diseases and/or symptoms. Wherein the food product comprises a health care product.

According to the invention, the treatment refers to an improvement or complete disappearance of a subject's symptoms associated with a disease or condition caused by miR-183, wherein a broad sense of improvement refers to a reduction of at least one parameter. Specific to the present application, for example, it may be an improvement in body weight, appetite, gastrointestinal digestive function and body fat, and the like.

The individual to be treated can be any individual, preferably a mammal, afflicted with the symptoms described above.

According to the present invention, the "inhibition of the function of miR-183" refers to the degree of down-regulation of miR-183 expression of its target gene in a target cell expressing miR-183 of the same species which is not treated by the method of the present invention, The miR-183-expressing target cells treated by the present invention have a reduced degree of down-regulation of their target gene expression by at least 0.5-fold, and can generally be reduced by at least a factor of two, such as Figures 2A and 2B.

The administration form and composition of the drug can be referred to the above description, and the present invention will not be described in detail herein.

The invention will be described in detail below by way of examples. In the following embodiments,

miR-183 overexpression vector

The (human) miR-183 gene (CCGCAGAGTGTGACTCCTGTTCTGTGTATGTCACTGGTAGAATTCACTGTGAAC AGTCTCAGTCAGTGAATTACCGAAGGGCCATA AACAGAGCAGAGACAGATCCACGA ) shown in SEQ ID No: 3 was cloned into the pCAG-GFP vector to obtain the overexpression plasmid pCAG-miR-183-GFP of the miR-183 gene. Among them, the synthesis and cloning of the miR-183 gene shown by SEQ ID No: 3 was carried out by Kingsray.

miR-183 sensor vector (miR-183 sensor vector)

The miR-183 receptor vector is a fire luciferase gene 3' cloned into the pGL3-SV40 vector by binding and modulating the target sequence (TGGAAATGAGATCTTGTGCCATAGCTACGGTAAGGATTTTCAGTGCCATT) shown in SEQ ID No: 11 to the two miR-183s. The downstream xbaI site was obtained, so that the expression of fire luciferase in the miR-183 receptor vector was regulated by miR-183.

Example 1

This example is used to illustrate the difference in miR-183 expression in 2 month old and 12 month old mice.

(1) Extraction and reverse transcription of total RNA

Wild type C57/Bl6 male mice, 2 months old and 12 months old, were sacrificed after cervical dislocation, 500 μl of blood was taken, and the entire hypothalamus and half stomach were dissected, and 200 mg of liver, muscle and epididymis were taken. Adipose tissue. Add 1 ml of Trizol reagent (invitrogen) to the blood and mix. Then add 200 μl of Trizol reagent to the hypothalamus, stomach, liver, muscle and adipose tissue, then cut the hypothalamus, stomach, liver, muscle and adipose tissue with scissors, and then grind these tissues into pieces with an electric homogenizer. Then, the total RNA in the whole tissue was extracted according to the instructions of Trizol. The RNA was solubilized with nuclease-free water, and then the ratio of 260 to 280 of the RNA was determined using a Nanodrop 2000 instrument, and samples with a ratio greater than 1.8 were continued for subsequent experiments. After the concentration of RNA was determined by Qubit, the integrity of the RNA was detected using a bioanalyzer, and the RNA integrity index RIN was greater than 0.9. Among them, in order to ensure that each tissue or organ can perform subsequent experiments, multiple replicates can be set for total RNA extraction.

1 μg of total RNA was taken from each sample, and 10-40 nt of short RNA in total RNA was isolated using a flashPAGE sieving apparatus (Ambion), and then a microRNA cDNA library was prepared by reverse transcription using an Illumina kit, and then in a second The expression level of microRNA in the sample was determined on a generation sequencer.

The results showed that the expression of miR-183 in the hypothalamus, stomach and liver of the 12-month-old mice was higher than that in the 2-month-old mice, indicating that the expression level of miR-183 increased with age in the hypothalamus. Increased in the stomach and liver, positively correlated with weight gain and obesity.

(2) Quantitative PCR detection of miR-183 expression

To confirm that the expression level of miR-183 in the hypothalamus, stomach and liver increased with age, the expression level of miR-183 was detected by quantitative PCR. 1 μg of total RNA was taken from each sample, and cDNAs of microRNAs and mRNAs were reverse transcribed using Catch AllTM miRNA & mRNA RT-PCR kit (Pengekiphen, Kunshan). The primers used for detection were: miR-183 forward primer (5'-TATGGCACTGGTAGAATTCACT-3') as shown in SEQ ID No: 12; U6 forward primer as shown in SEQ ID No: 13 (5'-CGCAAGGATGACACGCAAATTCG) -3'); The reverse primer is a universal primer provided for the kit. The instrument to be tested was Bio-Rad's iQ5 system and the reagent was TaKaRa's SYBR Green Mix. Three replicate wells were simultaneously detected for each sample, and U6 was used as an internal reference to calculate the expression level of miR-183 in each sample by the 2-ΔΔct method. The expression level of miR-183 in each organ of 2 month old mice was then set to 1, and the relative expression level of miR-183 in 12-month-old mice was calculated. The results are shown in Fig. 1.

As shown in Figure 1, the expression level of miR-183 in the hypothalamic-gastric central axis of the appetite was significantly up-regulated in 12-month-old mice, and the expression of miR-183 in the liver was also significantly up-regulated. Quantitative PCR confirmed that the expression levels of miR-183 in the hypothalamus, liver and stomach of the 12-month-old mice were 1.8, 1.6 and 2.4 times higher than those in the 2-month-old mice, respectively. It can be shown that up-regulation of miR-183 is associated with weight gain and fatty liver formation. Among them, in Fig. 1, **P<0.01, *p<0.05.

Example 2

This example is to illustrate the in vitro regulation of antisense oligonucleotides on miR-183

Human embryonic kidney cells HEK-293T were cultured in DMEM medium containing 10% fetal bovine serum. The cell culture incubator was constantly maintained at 37 ° C and 5% CO ₂ . HEK-293T cells were seeded in a 24-well cell culture plate at a seeding rate of 100,000 cells per well at a culture volume of 500 μl. The following day, the settings of Table 2 below were co-transfected into KEK-293 cells using liposome 2000 (Invitrogen) according to the instructions, and expression from the miR-183 receptor vector was measured 36 hours later using a dual luciferase analyzer (Promega). The luciferase activity. Three replicate wells were set each time and the experiment was repeated three times.

Among them, in each group, the amount of miR-183 receptor vector was transferred per well: 500 ng of miR-183 receptor vector, 20 ng of pCAG-GFP blank vector, 500 ng of miR-183 overexpression vector, and 50 μM of oligonucleotide The solution. Further, when the transferred oligonucleotide was miR-183 ASO, 0.5 μl and 1 μl of a 50 μM oligonucleotide solution were added, respectively, and the final concentrations were 0.05 μM and 0.1 μM after being added to the cell culture solution. To determine the activity of luciferase, and to take the ordinate as the ordinate, the miR-183 ASO concentration is plotted on the abscissa. The results are shown in Figure 2.

As can be seen from Figure 2, miR-183 ASO can inhibit the function of miR-183.

As can be seen from Fig. 2A, the miR-183 receptor vector, miR-183 overexpression vector and different concentrations of miR-183 ASO were co-transfected, and the luciferase activity assay showed that miR-183 ASO could inhibit the function of miR-183. And there is a dose effect.

As can be seen from Figure 2B, overexpression of miR-183 inhibits the expression of miR-183 receptor vector (left 1 column and left 2 column); miR-183 ASO and miR-183 mismatch ASO can inhibit the function of miR-183 ( The left 4 column and the left 5 column), the miR-183 ASO inhibition effect is better than the miR-183 mismatch ASO, and the random control nucleotide can not inhibit the miR-183 function (left 3 column), Data = average ± SEM; N = 3; *** P < 0.001, ** P < 0.01. Furthermore, overexpression of miR-183 in HEK293 cells inhibited the expression level of the reporter luciferase in the miR-9 receptor vector to 46% of the control group, while co-transforming the final concentration of 0.1 μM of miR-183 ASO The expression of the reporter gene luciferase in the miR-183 receptor vector was restored to 76% of the control level, that is, miR-183 ASO was able to inhibit 56% of miR-183 function.

Table 2

Example 3

This example is to demonstrate that the antisense oligonucleotide of miR-183 regulates the miR-183 target gene in vivo and its subsequent effects on appetite, gastrointestinal function and lipid metabolism.

Ten wild-type C57/Bl6 male mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) with 12-week-old body weight difference of less than 10% were randomly divided into two groups, after feeding for two weeks (feeding 20% high) Fat feed), mice in the experimental group were intragastrically administered with 8 mg/kg body weight of miR-183 ASO (dissolved in PBS), and control mice were intragastrically administrated with an equal volume of solvent PBS. The stomach was weighed once a week and the body weight and food consumption of the mice were weighed. Body weight changes and weight gain rates are shown in Figures 3A and 3B, respectively. The thickness of subcutaneous fat in the miR-183 ASO mice was also thicker than the control group.

As can be seen from Figure 3, the antisense oligonucleotide (miR-183 ASO) administered by miR-183 was able to promote weight gain in mice (Fig. 3A) and body weight growth rate compared with the control group (Fig. 3B). ). Data = mean ± SD; N = 5; * P < 0.05.

As can be seen from Figure 4, compared with the control group, gR-183 ASO can increase the food intake, **P<0.01.

As can be seen from Figure 5, gR-183 ASO can increase the weight of gastric and abdominal fat compared to the control group (Fig. 4); however, it has no effect on kidney weight. *P<0.05, **P<0.01.

The total RNA of the hypothalamus and stomach of the mice in the comparison experiment group and the miR-183 ASO gavage group were extracted according to the method in Example 1, and sequenced and analyzed by the second generation: miR-183 ASO compared with the comparison experiment group The gavage group up-regulated many genes involved in glycolipid metabolism or type 2 diabetes or liver disease-associated PI3K-Akt signaling pathway, MAPK signaling pathway, TGF-beta signaling pathway, and GnRH signaling pathway (Table 3). In addition, many genes in gastric acid secretion, salivary secretion, phosphatidylinositol signaling pathway, insulin signaling pathway and type 2 diabetes signaling pathway were up-regulated (Table 3).

table 3

In summary, inhibition of miR-183 function can promote appetite, stomach weight and body weight, and increase subcutaneous fat and abdominal cavity content. Moreover, the molecular experiments of the present invention demonstrate that miR-183 regulates many genes in the appetite control, digestion and absorption, and fat metabolism signaling pathways in vivo, which clarifies that miR-183 antisense nucleotides can promote appetite, enhance gastrointestinal function, and increase The molecular mechanism of body weight and fat content.

Example 4

This example is used to demonstrate the in vitro regulation of small interfering RNA (siRNA) on miR-183

Human embryonic kidney cells HEK-293T were cultured in DMEM medium containing 10% fetal bovine serum. 37 [deg.] C incubator and maintained constant 5% CO _2, the culture volume of 500μl. The next day, miR-183 siRNA (antisense strand SEQ ID No: 7: 5'AGACUGUUCACAGUGAAUUCU'3, sense strand SEQ ID No: 8: 5'AGAAUUCACUGUGAACAGUCU'3, Shanghai Jima was prepared according to the instructions using liposome 2000 (Invitrogen). Synthesis, wherein the structure of dTdT at the 3' end of SEQ ID No: 7 and SEQ ID No: 8) and random control RNA (antisense strand SEQ ID No: 9: 5'CGUGACACGUUCGGAGAA'3; sense strand SEQ ID No: 10:5'UUCUCCGAACGUGUCACGU'3, Shanghai Gemma synthesis, in which the structure of dTdT is present at the 3' end of SEQ ID No: 9 and SEQ ID No: 10), respectively, transfected into KEK-293 into cells, 36 After an hour, the cells were lysed with Trizol and total RNA was extracted, and then the expression amount of miR-183 was detected using the same quantitative PCR kit and primers of Example 1. Three replicate wells were set each time and the experiment was repeated three times. The results are shown in Figure 6, ***P < 0.01.

The results showed that miR-183 siRNA could down-regulate the expression of miR-183 by 72%, and thus the amount of miR-183 bound to the target gene of miR-183 was also down-regulated by 72%, thereby increasing the expression level of the target gene. It can be seen that the function of miR-183 can be successfully inhibited by RNA interference with the miR-183 precursor.

to sum up

The present invention can sufficiently inhibit miR-183 in a target cell expressing miR-183 by contacting a miR-183 inhibitor (including an antisense oligonucleotide and an interfering RNA) with a target cell expressing miR-183 in vivo or in vitro. Function (antisense oligonucleotide inhibits the binding of miR-183 to its target gene, interfering RNA can reduce the expression of miR-183, thereby inhibiting the function of miR-183), and can promote appetite when used for individual administration Invigorating gastrointestinal function, increasing body weight and fat content, that is, the miR-183 inhibitor (including antisense oligonucleotide and interfering RNA) provided by the present invention can effectively prevent and/or treat an increase in miR-183 The disease caused, for example, is a disease or symptom such as wasting, anorexia, weak gastrointestinal function or less subcutaneous fat, and can be used to maintain the skin, enhance skin elasticity and luster.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variants All fall within the scope of protection of the present invention.

It should be further noted that the specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not be further described in various possible combinations.

In addition, any combination of various embodiments of the invention may be made as long as it does not deviate from the idea of the invention, and it should be regarded as the disclosure of the invention.

references

1. Buhmann, H., C. W. le Roux, and M. Bueter, The gut-brain axis in obesity. Best Pract Res Clin Gastroenterol, 2014. 28(4): p. 559-71.

2. Wang, P.Y., et al., Upper intestinal lipids trigger a gut-brain-liver axis to regulate glucose production. Nature, 2008. 452 (7190): p. 1012-6.

3.Beraza, N. and C. Trautwein, The gut-brain-liver axis: a new option to treat obesity and diabetes? Hepatology, 2008.48(3): p.1011-3.

4. Schneeberger, M., et al., Hypothalamic miRNAs: emerging roles in energy balance control. Front Neurosci, 2015. 9: p.

5. Yan Dapeng, Zhan Lixing. Research progress in adipocyte differentiation and its regulation. Chinese Journal of Cell Biology 2010, 32(5): 690-695.

6.Gehart, H., et al., MAPK signalling in Cell metabolism: stress or wellness? EMBO Rep, 2010.11(11): p.834-40.

7. Maiuri, T., J. Ho, and V. Stabolic, Regulation of adipocyte differentiation by distinct subcellular pools of protein kinase B (PKB/Akt). J Biol Chem, 2010. 285(20): p. 15038-47.

8. Dooley, S. and P.ten Dijke, TGF-beta in progression of liver disease. Cell Tissue Res, 2012. 347(1): p.245-56.

9. Goldfarb, S. and F. N. Ziyadeh, TGF-beta: a crucial component of the pathogenesis of diabetic nephropathy. Trans Am Clin Climatol Assoc, 2001. 112: p. 27-32; discussion 33.

10.Zhang, Q., et al., Berberine moderates glucose metabolism through the GnRH-GLP-1 and MAPK pathways in the intestine. BMC Complement Altern Med, 2014.14: p.188.

Claims (12)

The use of inhibiting the function of miR-183 in increasing body weight, particularly in the preparation of drugs and/or foods for the prevention and/or treatment of wasting. The use of miR-183 to inhibit the appetite and gastrointestinal functions, particularly in the preparation of drugs and/or foods for the prevention and/or treatment of anorexia, dyspepsia or weak gastrointestinal functions. The use of inhibiting the function of miR-183 in increasing the fat content in subcutaneous fat and skin cells, especially in the preparation of skin care products for skin care, skin elasticity and gloss. The use according to any one of claims 1 to 3, wherein miR-183 has the nucleotide sequence shown in SEQ ID No: 1. A method of inhibiting the function of miR-183, the method comprising: contacting a miR-183 inhibitor with a target cell expressing miR-183. The method according to claim 5, wherein the miR-183 inhibitor is an antisense oligonucleotide comprising antisense DNA and antisense RNA, the antisense oligonucleotide is complementary to miR-183, and a sequence having a length of 8-40 nucleotides and having a nucleotide complementary to nucleotides 2-8 of miR-183; Preferably, the antisense oligonucleotide has the following nucleotide sequence: a) the nucleotide sequence shown in SEQ ID No: 4; b) a nucleotide sequence which is deleted, substituted or added with one or several nucleotides and which is complementary to miR-183 in the nucleotide sequence shown in SEQ ID No: 4; preferably, in SEQ ID No: 4 a nucleotide sequence which is deleted, substituted or added with one or several nucleotides and which is at least 60% complementary to miR-183 in the nucleotide sequence shown; more preferably, with positions 2-8 of miR-183 A nucleotide sequence having at most 3 nucleotides mismatched nucleotides; most preferably, the nucleotide sequence shown in SEQ ID No: 5. The method according to claim 5 or 6, wherein the miR-183 inhibitor is a small interfering RNA of a miR-183 precursor; Preferably, the miR-183 precursor has the sequence set forth in SEQ ID No: 2; Preferably, the miR-183 precursor has a small interfering RNA length of 15-30 bp; Preferably, the small interfering RNA of the miR-183 precursor has an antisense strand as set forth in SEQ ID No: 7 and a sense strand as set forth in SEQ ID No: 8. A miR-183 inhibitor, wherein the miR-183 inhibitor is the antisense oligonucleotide of claim 6 and/or the small interfering RNA of the miR-183 precursor of claim . A pharmaceutical composition comprising the miR-183 inhibitor of claim 8 and a pharmaceutically acceptable carrier. A kit, comprising the miR-183 inhibitor of claim 8, optionally further comprising a pharmaceutically acceptable carrier. The method according to any one of claims 5 to 7, the miR-183 inhibitor according to claim 8, the pharmaceutical composition according to claim 9 and/or the kit according to claim 10 inhibiting miR- Application in the function of 183; preferably in increasing body weight, enhancing appetite and gastrointestinal function, increasing fat content in the subcutaneous fat and skin cells; more preferably in preventing and/or treating wasting, anorexia, indigestion, weak gastrointestinal function, An application in at least one of skin care, skin elasticity and gloss. Use of the miR-183 inhibitor according to claim 8 or the pharmaceutical composition according to claim 9 for the preparation of a medicament for inhibiting the function of miR-183; preferably for the preparation of a medicament for preventing and/or treating wasting and anorexia , indigestion, drugs and/or foods with weak gastrointestinal function, or applications in skin care products for skin care, skin elasticity and luster.

Images