CN114788865B - Application of miR-378 as cholesterol steady-state regulation target point - Google Patents

Abstract

The present invention reveals that miR-378 is a regulatory target of cholesterol homeostasis, and miR-378 and its regulators can be used as a target for developing drugs for high cholesterol-related diseases (such as hypercholesterolemia, atherosclerosis) or low-cholesterol-related diseases new target.

Description

Application of miR-378 as a regulatory target for cholesterol homeostasis

technical field

The invention belongs to the technical field of molecular biology, and more specifically, the invention relates to the application of miR-378 as a cholesterol homeostasis regulation target.

Background technique

MicroRNA is a kind of small molecule non-coding RNA with a length of 19-24nt in eukaryotes, which has the function of regulating gene expression. MicroRNA is mostly synthesized in vivo, and the genes encoding miRNA in the genome are transcribed under the action of RNA polymerase to produce longer primary transcripts (pri-miRNA), and pri-miRNA is processed into microRNA with a stem-loop structure under the action of Drosha The precursor (pre-miRNA), the pre-miRNA is transported out of the nucleus by exportin-5, and is further cleaved by Dicer in the cytoplasm to generate double-stranded miRNA, and then the double-stranded miRNA is melted, and finally mature miRNA is generated. Mature miRNAs will enter the RNA-induced silencing complex (RISC), thereby causing degradation or translational repression of target gene mRNAs, and microRNAs (miRNAs) increase the stability of gene post-transcriptional regulatory networks.

Thyroid hormone (TH) mainly acts through thyroid hormone receptors (TR). There are mainly two types of thyroid hormone receptors, α and β, which are differentially expressed in adult tissues. TRβ1 is the major TR in the liver and is mainly responsible for the effect of TH on cholesterol metabolism. Mouse models with TRβ1 mutations exhibit defects in cholesterol homeostasis. Based on the understanding of the mechanism of action of TH, in order to avoid adverse side effects on cardiac function and the hypothalamus-pituitary-thyroid axis, great progress has been made in recent years in the development of TRβ-selective or liver-specific TH agonists for the treatment of hyperthyroidism. Cholesterolemia and prevention of atherosclerotic cardiovascular disease. One of the mechanisms by which TH lowers cholesterol is that it can promote the synthesis of bile acid (BA), thereby increasing the conversion of cholesterol to BA in the liver and eliminating cholesterol from the body. There are two main pathways for the synthesis of bile acids, one is the classic pathway that mainly synthesizes cholic acid (CA) through CYP7A1 and CYP8B1, and the other is the bypass pathway that mainly synthesizes deoxycholic acid (CDCA) through CYP27A1 and CYP7B1. It is worth noting that there is a certain difference in the composition of bile acids between humans and mice. In humans, the bile acids synthesized by the classical pathway account for the vast majority, but in mice, the bile acids synthesized by the bypass pathway can account for up to 100%. About fifty. In addition, deoxycholic acid synthesized by the bypass pathway in the mouse liver was converted into aMCA and bMCA under the catalysis of CYP2C70 enzymes. Bile acids synthesized in the liver enter the gallbladder and are conjugated with glycine and taurine in the gallbladder and stored in the gallbladder. After the body eats, bile acids are secreted by the gallbladder into the small intestine, and under the action of the intestinal flora in the small intestine, they are dissociated from amino acids and further metabolized into secondary bile acids to help digestion and absorption. Most of the bile acids in the small intestine will be reabsorbed into the blood and then transported back to the gallbladder for storage. A small part will be excreted with the feces, and the body will further synthesize new bile acids to replace the lost bile acids. Studies have reported that TH can directly promote the expression of CYP7A1 in the promoter region of CYP7A1 through TR binding, but at the same time, some studies have also found that TH can inhibit the expression level of CYP8B1. In addition, chip-seq analysis found that TH may also regulate other enzymes in the bile acid synthesis pathway, which also shows that TH plays an important role in maintaining BA synthesis and homeostasis.

Although there is some research on the regulation of cholesterol in this field, those skilled in the art have also found that when TH or its analogues are used as drugs to treat diseases such as hypercholesterolemia, there will be many side effects. At present, it is still difficult to find other alternative medicines in this field, and there is no effective way to solve this problem.

Therefore, the field still needs to further screen out useful new targets for regulating cholesterol, so as to obtain drugs that are really useful for regulating cholesterol based on this.

Contents of the invention

The purpose of the present invention is to provide the application of miR-378 as a regulation target of cholesterol homeostasis.

In the first aspect of the present invention, there is provided the use of miR-378 or its regulator in the preparation of a composition for regulating serum cholesterol level or bile acid level.

In a preferred example, the regulator is an upregulator, and the miR-378 or its upregulator is used to prepare a composition for lowering serum cholesterol levels or increasing bile acid levels; preferably, for the preparation of preventive, A composition for improving or treating diseases related to high cholesterol; more preferably, the diseases related to high cholesterol include hypercholesterolemia and atherosclerosis.

In another preferred example, the miR-378 or its up-regulator is also used to prepare a composition with the following functions: increasing the expression of CYP7B1, CYP8B1, and CYP27A1; or participating in TH's action on the bile acid synthesis pathway by regulating MAFG Regulate the network.

In another preferred example, the upregulator of miR-378 includes: miR-378 mimics, miR-378 agonists, miR-378 precursors, expression systems expressing miR-378, miR-378 expression stimulators ;

In another preferred example, the expression system includes (but not limited to): expression plasmid, host cell or virus; the virus includes adenovirus, adeno-associated virus, and lentivirus.

In another preferred example, the regulator is a down-regulator, and the down-regulator of miR-378 is used to prepare a composition that increases serum cholesterol levels or reduces bile acid levels; preferably, it is used for the preparation of preventive, Composition for improving or treating diseases related to low cholesterol.

In another preferred example, the down-regulator of miR-378 includes: substances that inhibit or prevent miR-378 from binding to its target gene site; preferably Antagomir of miR-378, a combination of miR-378 Antisense nucleic acid or siRNA of miR-378; preferably, the target gene is MAFG.

In another aspect of the present invention, there is provided a composition for preventing, improving or treating hypercholesterolemia-related diseases, said composition comprising an up-regulator of miR-378, which comprises an expression system expressing miR-378; preferably Preferably, the expression system for expressing miR-378 is an expression plasmid, virus or host cell containing the nucleotide sequence of 5'-AGGGCTCCTGACTCCAGGTCCTGTGTGTTACCTGAAATAGCACTGGACTTGGAGTCAGAAGGCCT-3' (SEQ ID NO: 2).

In another aspect of the present invention, there is provided a composition for preventing, improving or treating low-cholesterol-related diseases, said composition comprising miR-378 down-regulator, which comprises inhibiting or preventing miR-378 and its targeting Gene locus binding substance; preferably, it is antagomir of miR-378.

In another aspect of the present invention, there is provided a method of screening substances (including potential substances) that regulate serum cholesterol levels or bile acid levels, the method comprising: (1) treating a system expressing miR-378 with a candidate; and (2) Detect the expression of miR-378 in the system; wherein, if the candidate can be increased (preferably significantly increased, such as increased by more than 20%, preferably increased by more than 50%; more preferably increased by more than 80%) The expression of miR-378 indicates that the candidate is a substance that reduces serum cholesterol levels or increases bile acid levels; if the candidate can be reduced (preferably significantly reduced, such as increased by more than 20%, preferably reduced by more than 50%; (more preferably, reduce the expression of miR-378 by more than 80%), it indicates that the candidate is a substance that increases serum cholesterol level or decreases bile acid level.

In a preferred example, step (1) includes: in the test group, adding the candidate to the system expressing miR-378; and/or, step (2) includes: detecting the expression of miR-378 in the system of the test group expression, and compared with the control group, wherein the control group is a system expressing miR-378 without adding the candidate; if the expression of miR-378 in the test group is statistically higher (preferably significantly higher, Such as increasing more than 20%, preferably increasing more than 50%; more preferably increasing more than 80%) the control group, it shows that the candidate is a substance that reduces serum cholesterol levels or increases bile acid levels; if miR-378 in the test group The expression of the expression is statistically lower (preferably significantly lower, such as a reduction of more than 20%, preferably a reduction of more than 50%; more preferably a reduction of more than 80%) in the control group, which indicates that the candidate is to improve serum cholesterol levels or Substances that lower bile acid levels.

In another aspect of the present invention, a nucleic acid complex is provided, which includes miR-378 and MAFG gene (preferably its mRNA), and the miR-378 binds to the 3'UTR of the MAFG gene.

In another preferred example, the nucleic acid complex is used to screen substances (including potential substances) that regulate serum cholesterol levels by regulating the interaction between miR-378 and MAFG gene.

In another aspect of the present invention, there is provided a method of screening for substances (including potential substances) that regulate serum cholesterol levels or bile acid levels, said method comprising: (1) interacting the candidate with miR-378 and MAFG gene (2) detecting the effect of the candidate on the interaction between miR-378 and the MAFG gene; wherein, if the candidate can promote the interaction between miR-378 and the MAFG gene, it indicates that the candidate is down-regulating serum cholesterol levels or substances that increase bile acid levels (which can be used to prepare compositions for preventing, improving or treating high cholesterol-related diseases); if the candidate can weaken the interaction between miR-378 and MAFG gene, it indicates that the candidate is to increase serum cholesterol Level or down-regulate the level of bile acid (can be used to prepare the composition for preventing, improving or treating diseases related to low cholesterol).

In another preferred embodiment, in the test group, the candidate is added to the system in which miR-378 interacts with the MAFG gene; and/or, step (2) includes: detecting miR-378 and MAFG in the system of the test group Gene interaction situation, and compare with control group, wherein said control group is the system that does not add described candidate miR-378 and MAFG gene interaction; If miR-378 and MAFG gene interaction in test group If it is higher than (preferably significantly higher than, such as higher than 20%, preferably higher than 50%; more preferably higher than 80%) the control group, it indicates that the candidate is to down-regulate serum cholesterol levels or up-regulate bile acid levels If the interaction between miR-378 and MAFG gene in the test group is statistically weaker (preferably significantly weaker, such as attenuation of more than 20%, preferably attenuation of more than 50%; more preferably attenuation of more than 80%) the control group, it indicates that the candidate is a substance that up-regulates serum cholesterol levels or down-regulates bile acid levels.

In another preferred example, the system further includes: CYP7B1, CYP8B1, and CYP27A1; by observing changes in the expression of CYP7B1, CYP8B1, and CYP27A1, the effect of the candidate is determined; preferably, if CYP7B1, CYP8B1, and CYP27A1 If the expression of CYP7B1, CYP8B1 and CYP27A1 is significantly decreased, it indicates that the candidate is a substance that up-regulates MAFG and up-regulates serum cholesterol levels.

In another preferred example, in the screening method, the candidate can be: a macromolecule designed based on a gene or protein that affects the activity or function of MAFG and/or miR-378, or regulates its expression ( Such as peptides, antibodies, overexpression vectors or interfering molecules) or small molecules (such as compounds).

In another preferred example, in the screening method, the system is selected from the group consisting of: cell system (including cell culture system), subcellular system, solution system, tissue system, organ system or animal system.

In another preferred example, the screening method further includes: conducting further cell experiments and/or animal experiments on the obtained substances, so as to further select and determine from the candidates useful substances for regulating serum cholesterol levels or bile acid levels. substance.

In another preferred example, the serum cholesterol level includes: low-density lipoprotein cholesterol level, high-density lipoprotein cholesterol level, or serum total cholesterol level.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. MiR-378 is positively regulated by thyroid hormones. (a) Schematic diagram of the establishment of a hypothyroid hyperthyroid mouse model. (b) Microarray analysis of differentially expressed microRNAs after thyroid hormone treatment. (c) The relative expression level of miR-378 in MMI (methimazole) group and MMI+T3 (methimazole+triiodothyronine) group. (d) Relative expression level of miR-378 in primary mouse hepatocytes with T3 treatment concentration and treatment time.

Figure 2. Overexpression and suppression of miR-378 in the liver can reduce and increase serum cholesterol levels. (a-b) Relative levels of miR-378 in liver (a) and total cholesterol in serum (b) after injection of Ad-Ctrl and Ad-378. (c-d) Levels of LDL-cholesterol (c) and HDL-cholesterol (d) in mouse serum after injection of Ad-Ctrl and Ad-378. (e–f) Relative levels of miR-378 in liver (e) and total cholesterol in serum (f) after injection of Ant-Ctrl and Ant-378.

Figure 3. MiR-378 can regulate genes related to bile acid synthesis. (a) Go and KEGG analysis of differentially expressed genes after miR-378 overexpression in liver. (b-c) Expression levels of mRNA (b) and protein (c) of key enzymes in the bile acid synthesis signaling pathway in the liver of mice infected with Ad-Ctrl and Ad-378. (d-e) Expression levels of mRNA (d) and protein (e) of key enzymes in the bile acid synthesis signaling pathway in the liver of mice injected with Ant-Ctrl and Ant-378. (f) mRNA expression levels of cholesterol absorption (LDLR, PCSK9, SR-BI), de novo synthesis (HMGCR, SREBP2) and reverse transport (ABCG5, ABCG8) in mice infected with Ad-Ctrl and Ad-378 . (g-h) Expression levels of mRNA (g) and protein (h) of key enzymes in the bile acid synthesis signaling pathway in primary mouse hepatocytes infected with Ad-Ctrl and Ad-378. (i-j) mRNA (i) and protein (j) expression levels of key enzymes in the bile acid synthesis signaling pathway in primary mouse hepatocytes treated with Ant-Ctrl and Ant-378

Figure 4. Overexpression of miR-378 in the liver can promote the synthesis and excretion of bile acids. (a) The content of total bile acids in the gallbladder of mice infected with Ad-Ctrl and Ad-378 and the total amount of primary bile acids produced by the classical pathway and the alternative pathway. (b) Percentage of primary bile acids produced by the classical pathway and the alternative pathway to the total bile acid content in the gallbladder of mice infected with Ad-Ctrl and Ad-378. (c) The total bile acid content in the feces of mice infected with Ad-Ctrl and Ad-378 and the total amount of secondary bile acids produced by the classical pathway and the alternative pathway. (d) Percentage of secondary bile acids produced by the classical pathway and the bypass pathway in the gallbladder of mice infected with Ad-Ctrl and Ad-378 in the total bile acid content.

Figure 5. MAFG is the target gene of miR-378. (a) Schematic diagram of the seed sequence of miR-378 and the binding site of MAFG3'UTR in multiple species. (b) The fluorescence intensity of the HEK 293T cell line after co-transfection of the fluorescent reporter carrier containing MAFG-3'UTR with the control and AgomiR-378. (c) Fluorescence intensity of the HEK293T cell line co-transfected with the fluorescent reporter carrier containing MAFG-3'UTR with the miR-378 binding site mutated, the control and AgomiR-378. (d) MAFG protein levels in primary hepatocytes infected with Ad-Ctrl or Ad-378. (e) MAFG protein level in primary hepatocytes transfected with Ant-Ctrl or Ant-378. (f) MAFG protein level in liver of mice transfected with Ad-Ctrl or Ad-378.

Figure 6. Inhibition of MAFG in the liver can reduce the total cholesterol level in mouse serum. (a) MAFG protein level in the liver of mice infected with Ad-shCtrl and Ad-shMAFG. (b) Relative levels of total cholesterol in serum of mice infected with Ad-shCtrl and Ad-shMAFG. (c) Contents of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum of mice after Ad-shCtrl and Ad-shMAFG injection. (d-e) Expression levels of mRNA (d) and protein (e) of key enzymes in the bile acid synthesis signaling pathway in the liver of mice infected with Ad-shCtrl and Ad-shMAFG. (f-g) Expression levels of mRNA (f) and protein (g) of key enzymes in the bile acid synthesis signaling pathway in primary mouse hepatocytes infected with Ad-shCtrl and Ad-shMAFG.

Figure 7. Inhibition of MAFG in the liver can promote the synthesis and excretion of bile acids. (a) The content of total bile acids in the gallbladder of mice infected with Ad-shCtrl and Ad-shMAFG and the total amount of primary bile acids produced by the classical pathway and the alternative pathway. (b) Percentage of the primary bile acids produced by the classical pathway and the alternative pathway in the gallbladder of mice infected with Ad-shCtrl and Ad-shMAFG in the total bile acid content. (c) The total bile acid content in the feces of mice infected with Ad-shCtrl and Ad-shMAFG and the total amount of secondary bile acids produced by the classical pathway and the alternative pathway. (d) Percentage of secondary bile acids produced by the classical pathway and the bypass pathway in the gallbladder of mice infected with Ad-shCtrl and Ad-shMAFG in the total bile acid content.

Figure 8. MAFG mediates the regulatory effects of miR-378 on serum cholesterol levels and bile acid synthesis and excretion. (a) The protein level of MAFG in the liver of mice infected with Ad-Ctrl, Ad-378 or Ad-378 and Ad-MAFG. (b) Relative levels of total cholesterol in mouse serum after Ad-Ctrl, Ad-378 or Ad-378 and Ad-MAFG infected mice. (c) Levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum of mice infected with Ad-Ctrl, Ad-378 or Ad-378 and Ad-MAFG . (d-e) Expression levels of mRNA (d) and protein (e) of key enzymes in the bile acid synthesis signaling pathway in the liver of mice infected with Ad-Ctrl, Ad-378 or Ad-378 and Ad-MAFG. (f, h) Expression levels of mRNA (f) and protein (h) of key enzymes in the bile acid synthesis signaling pathway in primary mouse hepatocytes infected with Ad-Ctrl, Ad-378 or Ad-378 and Ad-MAFG. (g, i) Total bile acid content in the gallbladder (g) and feces (i) of mice infected with Ad-Ctrl, Ad-378 or Ad-378 and Ad-MAFG, and the primary bile produced by the main pathway and the bypass pathway total amount of acid.

Figure 9. Moderate overexpression of miR-378 in the liver is sufficient to reduce serum cholesterol levels and promote bile acid synthesis and excretion. (a) Expression levels of miR-378 in livers of wild-type mice and miR-378 transgenic mice. (b) Relative levels of total cholesterol in the serum of wild-type mice and miR-378 transgenic mice (c-d) mRNAs of key enzymes in the bile acid synthesis signaling pathway in livers of wild-type mice and miR-378 transgenic mice (c) and protein (d) expression levels. (e-f) Expression levels of mRNA (e) and protein (f) of key enzymes in the bile acid synthesis signaling pathway in primary hepatocytes of wild-type mice and miR-378 transgenic mice. (g) MAFG protein expression levels in wild-type mice and miR-378 transgenic mice liver (h-i) total bile acid content in gallbladder (h) and feces (i) of wild-type mice and miR-378 transgenic mice and The total amount of primary and secondary bile acids produced by the main pathway and the bypass pathway. (j) Relative levels of serum total cholesterol in wild-type and miR-378 transgenic mice fed high-fat diet containing cholesterol.

Figure 10. MiR-378 knockout mice can increase serum cholesterol levels and inhibit the synthesis and excretion of bile acids. (a) Expression levels of miR-378 in livers of wild-type mice and miR-378 knockout mice. (b) Relative levels of total cholesterol in the serum of wild-type mice and miR-378 knockout mice (c-d) mRNAs of key enzymes in the bile acid synthesis signaling pathway in livers of wild-type mice and miR-378 knockout mice ( c) and protein (d) expression levels. (g) Protein expression levels of MAFG in livers of wild-type mice and miR-378 knockout mice. (f-g) Expression levels of mRNA (f) and protein (g) of key enzymes in the bile acid synthesis signaling pathway in primary hepatocytes of wild-type mice and miR-378 knockout mice. (h-i) Total bile acid contents in the gallbladder (h) and feces (i) of wild-type and miR-378 knockout mice, and the total amount of primary and secondary bile acids produced by the primary and secondary pathways. (j) MMI treatment of wild-type mice and miR-378 knockout mice, and the relative levels of serum total cholesterol in wild-type mice and miR-378 knockout mice treated with T3 after MMI treatment.

Figure 11. miR-378 and TH play an antagonistic and synergistic role in the regulation of bile acid synthesis. (a) Total cholesterol levels in serum of MMI and MMI+T3 treated mice. (b) Levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum of MMI and MMI+T3-treated mice. (c-d) Expression levels of mRNA (c) and protein (d) of key enzymes in the bile acid synthesis signaling pathway in the liver of mice treated with MMI and MMI+T3. (e-f) The total bile acid content in the gallbladder (e) and feces (f) of MMI and MMI+T3-treated mice, as well as the total amount of primary and secondary bile acids produced by the classical pathway and the alternative pathway. (g) Schematic illustration of miR-378-mediated TH effect on BA synthesis pathway. (h-i) Protein (h) and mRNA (i) expression levels of key enzymes in the bile acid synthesis signaling pathway in human hepatocytes infected with Ad-Ctrl or Ad-378.

Detailed ways

The inventors are committed to the research of cholesterol homeostasis regulation, and found that miR-378 regulates the V-Maf avian myofascial fibrosarcoma oncogene homologue G (MAFG), and then regulates serum cholesterol level or bile acid level, increases miR-378 can significantly reduce serum cholesterol levels and increase bile acid levels, so it can be used as a new approach for the development of drugs for high cholesterol-related diseases (such as hypercholesterolemia, atherosclerosis) or low cholesterol-related diseases.

miR-378 and its therapeutic uses

In the present invention, described miR-378 is the small ribonucleic acid with the nucleotide sequence shown in SEQ ID NO:1:

5'-ACUGGACUUGGAGUCAGAAGG-3' (SEQ ID NO: 1).

The miR-378 can be isolated from cells, or can be obtained by artificial synthesis. Precursors of miR-378 are also included in the present invention.

The inventors have found that miR-378 in the liver is positively regulated by TH, and at the same time, transient overexpression of miR-378 in the liver can reduce serum cholesterol levels, and the expression levels of key genes for bile acid synthesis are significantly up-regulated. Total bile acids in feces and the amount of bile acids synthesized by the classical pathway and the alternative pathway were significantly increased. The inventors further constructed animals with liver-specific overexpression of miR-378, and also found that moderately overexpressing the level of miR-378 in the liver can reduce the total cholesterol level in serum, while the bile acid synthesis process in the liver The expression levels of key enzymes were also significantly increased, and the content of bile acids in the gallbladder and feces were significantly increased. Further research by the inventors shows that miR-378 regulates the synthesis, excretion and cholesterol homeostasis of bile acids mainly through its direct target gene MAFG. At the same time, the present invention also shows that miR-378 plays both synergistic and antagonistic roles in the process of TH regulating bile acid synthesis. The present invention discovers for the first time the role and molecular mechanism of miR-378 in lowering serum total cholesterol. The present invention can not only further increase the understanding of the molecular mechanism of TH regulating bile acid and cholesterol homeostasis, but also contribute to the treatment of hypercholesterolemia and atherosclerosis. Diseases such as sclerosis provide new targets and treatment options.

Based on the elaboration of the present invention, miR-378 is a new drug target closely related to cholesterol regulation or bile acid regulation. Various therapeutic means targeting miR-378 can be used as novel and effective means to prevent and treat high-cholesterol-related diseases (such as hypercholesterolemia, atherosclerosis, etc.) or low-cholesterol-related diseases in animals (especially humans).

Moreover, miR-378 can also be used as a target for drug screening to screen for drugs that regulate cholesterol or bile acids by increasing the expression or activity of miR-378, thereby preventing, alleviating or treating related diseases.

Therapeutic uses of miR-378 modulators

Based on the inventor's new discovery, the up-regulator of miR-378 can be used to prepare a preparation for regulating cholesterol or bile acid, which can be a pharmaceutical composition, a health product composition or a food composition.

The upregulator of miR-378 refers to any agent that can increase the activity of miR-378 or its precursor, improve the stability of miR-378 or its precursor, up-regulate the expression of miR-378 or its precursor, increase miR- 378 or its precursor effective time, these substances can be used in the present invention, as a useful substance for up-regulating miR-378, thus can be used to regulate cholesterol or bile acid. They can be chemical compounds, small chemical molecules, biological molecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

As an option of the present invention, the miR-378 up-regulator is a miR-378 mimic (mimics) or agonist (Agomir), which has the same effect as the miR-378, or can increase the miR-378 Expression or activity of -378.

The up-regulator of miR-378 may be a modified up-regulator, and the modification includes: methoxylation modification, thiolation modification, cholesterol modification, alkyl modification, locked nucleic acid modification, peptide nucleic acid modification, and/or Or the antisense nucleotide whose phosphate backbone is replaced by a phospholipid link; preferably, the modification includes: cholesterol modification at the 3' end, two thio-skeleton modifications at the 5' end, and four thio-skeleton modifications at the 3' end , full-chain methoxy modification.

As a preferred option of the present invention, the miR-378 up-regulator is an expression system expressing miR-378. The expression system is not particularly limited, and any expression system capable of expressing activities similar to miR-378 or its analogs and mimics can be applied in the present invention. For example, the expression system includes, but is not limited to: expression plasmids, host cells containing expression plasmids, or viruses; the viruses include adenoviruses, adeno-associated viruses, lentiviruses, and the like. Those skilled in the art know how to express miR-378 or its analogs and mimics, and there are already some commercialized expression systems, which can all be applied in the present invention.

As used herein, the "miR-378 down-regulator" includes nucleic acid inhibitors, antagonists, inhibitors, blockers, blockers, etc., as long as they can down-regulate the expression level of miR-378 or its precursor. They can be chemical compounds, small chemical molecules, biological molecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

The miR-378 downregulator refers to any agent that can prevent miR-378 (especially the binding key site) or its precursor from binding to its target sequence, reduce the activity of miR-378 or its precursor, reduce miR- The stability of miR-378 or its precursor, down-regulating the expression of miR-378 or its precursor, reducing the effective time of miR-378 or its precursor, these substances can be used in the present invention, as useful for down-regulating miR-378 Substances that can be used to raise cholesterol levels or lower bile acid levels. For example, the inhibitors are: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, nucleases, nucleic acid binding molecules, as long as they can down-regulate the expression of miR-378. For example, the inhibitor is a nucleic acid inhibitor, which works by preventing miRNA from binding to its target sequence. The nucleic acid inhibitor is selected from (but not limited to): miRNA antisense nucleic acid, miRNA locked nucleic acid antisense nucleic acid; peptide nucleic acid; siRNA (silencing miRNA precursor).

pharmaceutical composition

The present invention also provides a composition, which contains the miR-378 or its modulator ( Including up-regulator or down-regulator), and pharmaceutically acceptable carrier. The compositions are useful for modulating cholesterol levels or bile acid levels.

As used herein, the "effective amount" refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in compositions may contain liquids, such as water, saline, buffers. In addition, there may also be auxiliary substances in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and the like. The carrier may also contain cell transfection reagents.

After knowing the use of the miR-378 or its modulator, various methods well known in the art can be used to administer the miR-378 or its modulator or their pharmaceutical composition to mammals. Including but not limited to: subcutaneous injection, intramuscular injection, intravenous infusion, transdermal administration, local administration, implantation, sustained release administration, etc.

The effective amount of miR-378 or its modulator described in the present invention can vary with the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the miR-378 or its regulator such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, route of administration, etc. Usually, when the miR-378 or its modulator of the present invention is administered at a daily dose of about 0.00001 mg-50 mg/kg animal body weight (preferably 0.0001 mg-10 mg/kg animal body weight), satisfactory effects can be obtained. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.

drug screening

After knowing the close correlation between miR-378 and cholesterol regulation or bile acid regulation, substances that regulate the expression of miR-378 (and regulators of miR-378) can be screened based on this feature.

Therefore, the present invention provides a method of screening substances that regulate cholesterol levels or bile acid levels, said method comprising: treating a system expressing miR-378 with a candidate; and detecting the expression of miR-378 in said system; if If the candidate can increase the expression of miR-378, it indicates that the candidate is a substance that lowers cholesterol or increases bile acid; otherwise, it indicates that the candidate is a substance that increases cholesterol or decreases bile acid. The system for expressing miR-378 may be, for example, a cell (or cell culture) system, and the cells may be endogenously expressing miR-378; or may be recombinantly expressing miR-378. The system for expressing miR-378 can also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, a rabbit, a sheep, a monkey, etc. )wait. In a preferred mode of the present invention, when screening, in order to more easily observe changes in the expression of miR-378, a control group can also be set, and the control group can be the expression of miR-378 without adding the candidate. system.

The present invention also provides a method for screening substances that regulate cholesterol levels or bile levels, the method comprising: (1) treating a system in which miR-378 interacts with the MAFG gene with a candidate; and (2) detecting the miR The interaction between miR-378 and MAFG gene in the system where -378 interacts with MAFG gene; if the candidate can promote the interaction between miR-378 and MAFG gene, it indicates that the candidate is a substance that lowers cholesterol levels and can be used for Prepare a composition for preventing, improving or treating high cholesterol-related diseases; if the candidate can weaken the interaction between miR-378 and MAFG gene, it indicates that the candidate is a substance that raises cholesterol levels, and can be used for the preparation of prevention, improvement or treatment Compositions for low cholesterol-associated disorders. In a preferred mode of the present invention, when screening, in order to more easily observe changes in the interaction between miR-378 and the MAFG gene, a control group can also be set, and the control group can be without adding the candidate The interaction system of miR-378 and MAFG gene.

As a preferred mode of the present invention, the method further includes: conducting further cell experiments and/or animal experiments on the obtained substances to further select and determine substances that are really useful for regulating cholesterol levels or bile acid levels.

The present invention has no special limitation on the detection method of the expression, activity and amount of miR-378 or MAFG. Conventional gene quantitative or semi-quantitative detection techniques can be used, such as (but not limited to): polymerase chain reaction (PCR), Northern blotting and the like.

On the other hand, the present invention also provides substances that can regulate cholesterol or bile acid levels obtained by the screening method. These initially screened substances can constitute a screening library, so that people can finally screen out substances that can regulate the expression and activity of miR-378, and then regulate the regulation of cholesterol levels or bile acid levels.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to the conditions described in J. Sambrook et al., edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer suggested conditions.

general method

a. Mice

The mouse C57BL/6J used in the experiment was purchased from Shanghai Slack Experimental Animal Company, and the db/db, ob/ob and control mice were purchased from the Institute of Model Biology, Nanjing University. The animals were kept at a constant temperature of 22-23°C in an SPF grade animal room, with a 12-hour day and night cycle, and were fed with normal feed. Adenovirus-mediated gene function studies were performed by tail vein injection.

b. Construction of adenovirus

The adenoviral overexpression system was constructed using AdEasy ^TM Adenoviral Vector System of Statagene Company. Expression sequences were cloned in the shuttle plasmids pShuttle-CMV or pAdTrack-CMV. After the shuttle plasmid was linearized with PmeI, it was transformed into BJ5183 competent cells, which were pre-transformed into the pAdEasy-1 plasmid. Recombinant plasmids were detected by picking small clones. The recombinant plasmid was digested with PacI and then transfected into 293A cells. When 90% of the cells showed CPE (cytopathic effect), the cells and culture medium were collected. Freezing and thawing three times releases virus particles, which is the P1 generation virus.

The expression sequence of the adenovirus (Ad-378) overexpressing miR-378 is: 5'-AGGGCTCCTGACTCCAGGTCCTGTGTGTTACCTAGAAATAGC ACTGGACTTGGAGTCAGAAGG CCT-3' (SEQ ID NO: 2);

Construction method of adenovirus (Ad-378) overexpressing miR-378: produced by using BLOCK-iTTMAdenovial RNAi Expression System of Invitrogen Company. The expression sequence (5'-AGGGCTCCTGACTCCAGGTCCTGTGTGTTACCTAGAAATAGC ACTGGACTTGGAGTCAGAAGG CCT-3'(SEQ ID NO: 2) was cloned on the pENTR-U6 vector, and pENTR-U6 and pAd/BLOCK-iT-DEST were recombined under the action of recombinase in the in vitro system into The new plasmid was then transfected into 293A cells after PacI linearization to obtain the P1 generation adenovirus. Further purification was performed to obtain purified virus.

The sequence of Antagomir (ANT-378) inhibiting miR-378 is: 5'-CCUUCUGACUCCAAAGUCCAGU-3' (SEQ ID NO:3).

The expression sequence of Ant-Ctrl is: 5'-UCUACUCUUUCUAGGAGGUUGUGA-3' (SEQ ID NO: 4).

d. Extraction of RNA

Extraction of RNA was performed according to a conventional method. Analysis of experimental results: after the reaction, confirm the amplification curve of Real Time PCR. Relative quantification was performed using ΔCt.

f. Western blot analysis

Western blot analysis was performed according to conventional methods.

g. Determination of serum cholesterol

Serum total cholesterol was measured using Wako's TCH detection kit. Low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined by kits from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

h. Detection of bile acid components

Bile acid components were detected by the mass spectrometry platform of Tianjin Medical University. Bile acids were extracted in 70% ethanol at 55°C for 4 hours, and then bile salts were analyzed by ultrafast liquid chromatography (UFLC)-triple time-of-flight/mass spectrometry (MS). The total BA content in gallbladder bile and feces was calculated by adding the values for the various bile acid species.

Example 1, MiR-378 is positively regulated by thyroid hormone

The inventors established animal models of hypothyroidism and hyperthyroidism by adopting the classical hypothyroidism and hyperthyroidism mouse model ( FIG. 1 a ). On this basis, the microRNA array chip of Exiqon Company was used to analyze the miRNA expression profile in the liver of hyperthyroidism and hypothyroidism mice. From many miRNAs with obvious differences, the inventor determined miR-378 as the research object after extensive analysis , which was significantly up-regulated in the liver of hyperthyroid mice (Fig. 1b).

Further verification by QPCR found that the expression level of miR-378 in the liver of mice treated with T3 (triiodothyronine) was significantly increased (Figure 1c), and the primary cultured mouse hepatocytes in vitro were also detected To the same change trend (Figure 1d).

Example 2. Overexpression of miR-378 in the liver can reduce serum total cholesterol levels and promote bile acid synthesis

(1) miR-378 regulates serum cholesterol levels

In order to explore whether miR-378 can affect the cholesterol metabolism in mice, the inventors further constructed an adenovirus (Ad-378) overexpressing miR-378 (Fig. 2a), and infected the liver of mice overexpressing miR- After 378, a significant decrease in serum cholesterol levels could be detected (Fig. 2b). At the same time, the inventors also found that after Ad-378 infection, the levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in the serum of mice were significantly decreased (Fig. 2c-d).

After inhibition of miR-378 with antagomir against miR-378 (ANT-378) (Fig. 2e), a significant increase in serum cholesterol levels could be detected (Fig. 2f).

These findings suggest that miR-378 in the liver can effectively regulate serum cholesterol levels.

(2) miR-378 is involved in the synthesis of bile acids in the liver

The metabolic network of cholesterol in the liver can be roughly divided into cholesterol absorption, de novo synthesis, reverse transport, and metabolism to synthesize bile acids. The inventors further used RNA-seq to detect the differentially expressed genes in the mouse liver after miR-378 was overexpressed, and after analyzing the KEGG signaling pathway, it was found that the gene expression level in the signaling pathway of primary bile acid synthesis in the liver was significantly increased ( Figure 3a). Real-time quantitative PCR was used to detect the RNA (Fig. 3b, d) and protein expression levels (Fig. , e) were significantly increased, but the RNA and protein expression levels of the key enzyme CYP7A1 in the classic pathway of bile acid synthesis did not change significantly (Fig. 3b-e). At the same time, it was found that after miR-378 was overexpressed in the liver, there were no significant changes in the genes involved in cholesterol absorption (LDLR, PCSK9, SR-BI), de novo synthesis (HMGCR, SREBP2) and reverse transport (ABCG5, ABCG8) ( Figure 3f). At the same time, the inventors also detected consistent changes in primary liver cells of mice (Fig. 3g-j). These results suggest that miR-378 can effectively promote the synthesis of bile acids in the liver and reduce the cholesterol level in serum.

The inventors further used chromatography-mass spectrometry to detect the levels of bile acids in the gallbladder and feces, and found that after overexpressing miR-378 in the liver, the total bile acids (BAs) in the gallbladder, the bile acids synthesized by the classical pathway (CAs including CA, TCA, GCA) and the total amount of bile acids synthesized by the bypass pathway (MCAs including MCA, TMCA) all increased significantly (Fig. 4a); but the content of CAs and MCAs in the total bile acids did not change significantly (Fig. 4b), indicating that miR-378 can simultaneously affect the classic pathway of bile acid synthesis and the selective pathway to increase the synthesis of bile acid, but it does not affect the content of bile acid components in the gallbladder. Similarly, after overexpression of miR-378 in the liver, total bile acids (BAs) in feces and secondary bile acids synthesized by the classical pathway (Cas including CA, DCA) and secondary bile acids synthesized by the alternative pathway (MCAs including aMCA, The total amount of bMCA) was significantly increased (Figure 4c), but the content of CAs and MCAs in total bile acids did not change significantly (Figure 4d), indicating that miR-378 in the liver can promote the excretion of bile acids with feces.

The above results suggest that miR-378 can effectively promote the synthesis and excretion of bile acids and reduce the cholesterol level in serum.

Example 3, MAFG is the target gene of miR-378

In order to further explore the molecular mechanism of miR-378 regulating cholesterol and bile acid metabolism, the inventors screened the genes involved in cholesterol and bile acid metabolism targeted by miR-378, and found that the 3'UTR of MAFG contains the height of miR-378. Conserved binding site (Fig. 5a, inside the dotted box). Thus, miR-378 is targeted to bind to the 3'UTR of MAFG.

For further verification, the inventors cloned the 3'UTR sequence of MAFG containing the miR-378 binding site (Figure 5a) onto a fluorescent reporter gene (pRL-TK plasmid), and co-transfected into 293T cells together with AgomiR-378 , the inventors found that overexpression of miR-378 could inhibit the fluorescence intensity of MAFG-3'UTR (Fig. 5b). However, when the binding site of miR-378 on MAFG-3'UTR was mutated, the inhibitory effect of miR-378 on the fluorescence intensity of MAFG-3'UTR disappeared (Figure 5c).

Mut1: Mutate the 5'-AGUC C AG-3' segment to 5'-AGUC G AG-3';

Mut2: Mutate the 5'-AGU CC AG-3' segment to 5'-AGU GG AG-3'.

The inventors further found in primary liver cells of mice that overexpression and inhibition of miR-378 could significantly reduce and increase the protein level of MAFG (Fig. 5d-e). Likewise, the protein level of MAFG was also significantly decreased after miR-378 was overexpressed in the liver (Fig. 5f).

These results indicated that MAFG is a target gene of miR-378.

Example 4. Inhibition of MAFG in the liver can reduce serum cholesterol levels and promote the synthesis and excretion of bile acids

Although MAFG was found to inhibit bile acid synthesis, whether MAFG can affect cholesterol metabolism still needs further exploration. The present inventors constructed an shRNA plasmid against MAFG and further constructed an adenovirus ( FIG. 6 a ).

Using pENTR-U6 as the backbone plasmid, the shRNA sequence inserted into the plasmid is:

5'-GCCACCAGCGTCATCACAATACGAATATTGTGATGACGCTGGTGGC-3' (SEQ ID NO: 5).

The inventors found that after inhibiting the expression of MAFG in the liver, the serum cholesterol level decreased significantly (Fig. 6b), and the serum low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) The levels were significantly decreased (Fig. 6C), indicating that MAFG may mediate the regulatory effect of miR-378 on cholesterol. The inventors further found that after knocking down MAFG in the liver, the RNA (Fig. 6d) and protein (Fig. 6e) expression levels of CYP7B1, CYP8B1 and CYP27A1 were all significantly increased, but the expression level of CYP7A1 was not significantly changed (Fig. 6d-e ). Consistent with this, the inventors also found that the RNA (Figure 6f) and protein (Figure 6g) expression levels of CYP7B1, CYP8B1 and CYP27A1 were significantly increased after inhibiting the expression level of MAFG in primary hepatocytes, but the expression level of CYP7A1 was not. Significant changes (Fig. 6f-g). This is consistent with the overexpression of miR-378 in the liver. These results suggest that overexpression of miR-378 in the liver reduces serum cholesterol levels by inhibiting MAFG expression.

The present inventors found that after inhibiting the expression level of MAFG in the liver, the total amount of BAs, CAs and MCAs in the gallbladder was significantly increased (Fig. 7a), indicating that inhibiting MAFG in the liver can promote both the canonical pathway and selective pathway of bile acid synthesis bile acid synthesis in the sexual pathway. At the same time, the ratios of CAs and MCAs to total bile acids in the gallbladder tended to increase and decrease, respectively (Fig. 7b). Similarly, the inventors also found that the total amount of BAs, CAs and MCAs increased significantly in the feces of mice (Fig. 7c), but the percentages of CAs and MCAs did not change significantly (Fig. 7d).

These results further suggest that miR-378 can effectively inhibit MAFG and promote the synthesis and excretion of bile acids.

Example 5, MAFG mediates the effects of miR-378 on serum cholesterol and bile acid synthesis and excretion

In order to further explore whether MAFG mediates the regulation of miR-378 on serum cholesterol and bile acid, the inventors restored the expression level of MAFG in the liver of mice overexpressing miR-378 (Figure 8a), and found that the expression level of MAFG was restored. After expression levels, miR-378 was able to counteract the effects of miR-378 on serum cholesterol levels (Fig. 8b) high LDL cholesterol levels (Fig. 8c) as well as bile acid synthesis genes (CYP7B1, CYP8B1 and CYP27A1) mRNA (Fig. 8d) and protein (Fig. 8e ) expression level. Restoring the expression level of MAFG in primary hepatocytes of mice can also counteract the effect of miR-378 on the expression levels of bile acid synthesis genes (CYP7B1, CYP8B1 and CYP27A1) mRNA (Figure 8f) and protein (Figure 8h). Similarly, Restoring the expression level of MAFG in the liver attenuated the effect of Ad-378 on the total amount of BAs, CAs and MCAs in gallbladder (Fig. 8g) and feces (Fig. 8i).

Based on the above results, MAFG is the main mediator molecule of miR-378 in the regulation of cholesterol and bile acid metabolism.

Example 6. Overexpression of miR-378 in the liver can reduce serum cholesterol levels and promote the synthesis and excretion of bile acids

Due to the overexpression of miR-378 in the liver by Ad-378 is exogenous intervention and the expression fold is higher. In order to further explore whether the overexpression of miR-378 in the liver can regulate cholesterol and bile acid metabolism under physiological conditions, the inventors constructed liver-specific overexpression mice (Fig. 9a). Construction method: Shanghai Southern Model Animal Center constructed miR-378 transgenic mice. miR-378* and miR-378 are contained in the same microRNA precursor sequence. This invention studies miR-378, adenovirus and transgenic mice Both overexpressed their precursor sequences. Specifically, the pA-EGFP-loxp-loxp2272-EF1a-loxp-loxp2272-miR-378/miR-378*-pA expression cassette was inserted into the Rosa26 gene locus through ES cells. The initial orientation of the EF1a promoter does not initiate transcription of the downstream target gene miR-378/378*. These transgenic mice were mated with mice expressing liver-specific cre enzyme (Alb-Cre) to obtain liver-specific miR-378/378* transgenic mice.

Surprisingly, the inventors found that after miR-378 was overexpressed in the liver, the serum cholesterol level of mice could also be significantly decreased (Fig. 9b). At the same time, the expression levels of RNA (Figure 9c) and protein (Figure 9d) of bile acid synthesis genes (CYP7B1, CYP8B1 and CYP27A1) in the liver were also significantly increased. Correspondingly, the expression levels of mRNA (Figure 9e) and protein (Figure 9f) of bile acid synthesis genes (CYP7B1, CYP8B1 and CYP27A1) in primary hepatocytes of transgenic mice were also significantly increased compared with wild type. At the same time, it was also found that the protein expression level of MAFG in the liver of the transgenic mice was significantly decreased, which was consistent with the previous research results of the present inventors ( FIG. 9 g ). The inventors also found that the total amount of BAs, CAs and MCAs in the gallbladder (Fig. 9h) and feces (Fig. 9i) of transgenic mice also increased significantly. The above results indicated that moderate overexpression of miR-378 in the liver was sufficient to suppress the expression level of MAFG, increase bile acid synthesis and reduce serum cholesterol.

At the same time, the inventors also used high-cholesterol food to feed wild-type and transgenic mice and found that the transgenic mice could resist hypercholesterolemia caused by high-cholesterol food (Figure 9j), which indicated that miR-378 in the liver could become Targets for the treatment of hypercholesterolemia.

Example 7. Knocking out miR-378 can increase serum cholesterol levels and inhibit the synthesis and excretion of bile acids

The present inventors further analyzed miR-378KO mice (Fig. 10a), and the miR-378/378* gene knockout mice (378KO) were provided by Researcher Yan Jun from the Institute of Model Animals, Nanjing University.

It was found that after miR-378 was knocked out in mice, the serum cholesterol level of the mice increased significantly (Fig. 10b). Further studies showed that the RNA (Figure 10c) and protein (Figure 10d) expression levels of bile acid synthesis genes (CYP7B1, CYP8B1 and CYP27A1) in the liver were also significantly decreased after miR-378 knockout. The protein level of MAFG was significantly increased in the liver of knockout mice (Fig. 10e). At the same time, the expression levels of mRNA (Figure 10f) and protein (Figure 10g) of bile acid synthesis genes (CYP7B1, CYP8B1 and CYP27A1) in primary hepatocytes of miR-378 knockout mice were also significantly lower than those of wild type. The present inventors also found that the total amount of BAs, CAs and MCAs in gallbladder (Fig. 10h) and feces (Fig. 10i) of miR-378 knockout mice were significantly decreased. The inventors used MMI (methimazole) and MMI+T3 to treat wild-type and miR-378 knockout mice and found that the responsiveness of miR-378 knockout mice to T3 was significantly lower than that of wild-type mice (Fig. 10j), which also suggests that miR-378 plays an important mediating role in the regulation of cholesterol levels by T3.

The above results indicated that knocking out miR-378 in mice could increase serum cholesterol levels in mice and inhibit the synthesis and excretion of bile acids.

Example 8. miR-378 and TH play an antagonistic and synergistic role in the regulation of bile acid synthesis

Consistent with the results of overexpressing miR-378 in the liver, the inventors found that T3 was able to reduce serum total cholesterol levels (Fig. 11a) as well as LDL and HDL levels (Fig. 11b). Studies have reported that TH can regulate bile acid synthesis. The inventors found that T3 was able to promote CYP7A1, CYP7B1 and CYP27A1, but inhibit the expression level of CYP8B1 (Fig. 11c-d). The inventors analyzed the bile acids in the mouse gallbladder and feces and found that T3 can also increase bile acid synthesis ( FIG. 11 e ) and excretion ( FIG. 11 f ). The inventor's research found that miR-378 participates in the regulatory network of TH on the bile acid synthesis pathway through its target gene MAFG to promote CYP8B1 and promote CYP7B1 and CYP27A1, and it plays an antagonistic and synergistic role with TH in regulating bile acid synthesis. (Fig. 11g).

Because there is a certain difference in the metabolism of bile acids between humans and mice, the inventors further used human pluripotent stem cells to differentiate into hepatocytes, and treated them with Ad-378, and found that miR- After 378 hours, it can promote the expression levels of RNA (Figure 11h) and protein (Figure 11i) of bile acid synthesis genes (CYP7B1, CYP8B1 and CYP27A1), but does not affect the expression of CYP7A1. And the protein level of MAFG decreased significantly (Fig. 11h). This indicates that miR-378 and its target gene MAFG have the potential to be drug targets for the treatment of human hypercholesterolemia.

Embodiment 9, drug screening

1. Drug screening with cells expressing miR-378

Primary cultured hepatocytes from mice can express miR-378 endogenously. This cell was used as a cell model for screening for regulation of cholesterol levels.

Test group: the above-mentioned cells are treated with the candidate;

Control group: the above cells were not treated with the candidate.

After treatment, the cells were assayed for miR-378 expression. If the expression of miR-378 in the test group is significantly increased compared with the control group, it indicates that the candidate is a substance potentially useful for lowering cholesterol level or increasing bile acid.

Transfection of mouse primary cultured hepatocytes with Ad-378 obtained above can increase the expression of intracellular miR-378, so miR-378 is a useful substance for lowering cholesterol levels.

2. Drug screening based on the interaction between MAFG and miR-378

Primary cultured hepatocytes from mice can express MAFG and miR-378 endogenously. This cell was used as a cell model for screening for regulation of cholesterol levels.

Test group: hepatocytes expressing MAFG and miR-378 were treated with the candidate;

Control group: hepatocytes expressing MAFG and miR-378 not treated with candidate.

After treatment, the effect of miR-378 on MAFG in the cells was observed. If the inhibitory effect of miR-378 on MAFG is significantly increased in the test group compared with the control group, it indicates that the candidate is potentially useful for lowering cholesterol levels.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

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Claims (5)

1. Use of a mir-378 regulator in the manufacture of a medicament for modulating serum cholesterol levels or bile acid levels;

   the regulator is an up regulator, and is used for preparing medicines for reducing serum cholesterol level or improving bile acid level; the up regulator is an expression system for expressing miR-378, and the expression system is an expression plasmid, a host cell or a virus; or (b)

   The regulator is a down regulator for preparing medicines for increasing serum cholesterol level or reducing bile acid level; the downregulator is selected from: antagomir of miR-378, antisense nucleic acid or siRNA of miR-378; the nucleotide sequence of the Antagomir is shown as SEQ ID NO. 3.
2. 2. The use of claim 1, wherein the miR-378 upregulation is used in the manufacture of a medicament for the prevention or treatment of hypercholesterolemia; the high cholesterol disease is as follows: hypercholesterolemia, atherosclerosis.
3. 3. The use of claim 1, wherein the miR-378 upregulation increases expression of CYP7B1, CYP8B1, CYP27 A1; or by regulating MAFG, participate in the regulation network of TH to bile acid synthesis pathway.
4. 4. The use according to claim 1, wherein the virus is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus.
5. 5. Application of miR-378 expression system in preparation of medicines for preventing or treating high cholesterol diseases, wherein miR-378 expression system is an expression plasmid, virus or host cell containing 5'-AGGGCTCCTGACTCCAGGTCCTGTGTGTTACCTAGAAATAGCACTGGAC TTGGAGTCAGAAGGCCT-3' nucleotide sequence; the high cholesterol disease is as follows: hypercholesterolemia, atherosclerosis.