CN114272257B

CN114272257B - Application of miR-204-5p mimics or miR-204-3p mimics in preparation of drugs for treating atherosclerosis

Info

Publication number: CN114272257B
Application number: CN202210033057.6A
Authority: CN
Inventors: 梁思佳; 刘秀
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2022-01-12
Filing date: 2022-01-12
Publication date: 2022-11-04
Anticipated expiration: 2042-01-12
Also published as: CN114272257A

Abstract

The invention relates to the field of cardiovascular diseases, in particular to application of miR-204 in diagnosis or treatment of atherosclerosis. The invention discovers for the first time that miR-204 has the effect of 'one-stone-two-bird', two mature bodies thereof, namely miR-204-5p and miR-204-3p inhibit the expression of scavenger receptors in cytoplasm and nucleus through different mechanisms respectively, miR-204-5p in cytoplasm inhibits SR-A expression through classical seed sequence targeting, and miR-204-3p inhibits the enrichment of H3K27ac and H3K4me3 on CD36 promoters in nucleus so as to inhibit the transcription of CD 36. miR-204 inhibits macrophage foaming and atherosclerosis through the above ways and under double-pipe conditions.

Description

Application of miR-204-5p mimics or miR-204-3p mimics in preparation of drugs for treating atherosclerosis

Technical Field

The invention relates to the field of cardiovascular diseases, in particular to application of miR-204 in diagnosis or treatment of atherosclerosis.

Background

Atherosclerosis (AS) is a common chronic metabolic cardiovascular and cerebrovascular disease and also a pathological basis of a plurality of cardiovascular and cerebrovascular diseases, and acute myocardial infarction and stroke caused by AS have extremely high lethality, and become one of the main diseases threatening human health. Therefore, the prevention and treatment of AS has been one of the hot spots of basic and clinical research.

The etiology and pathogenesis of AS are very complex, and its formation is through a series of complex processes, mainly including foaming of macrophages, deposition of lipids under endothelium, infiltration of inflammatory cells under endothelium, formation of lipid necrosis core, proliferation and migration of vascular smooth muscle cells, etc. AS is formed first in the intima layer of blood vessels, in the early stage of the disease course of AS, under various pathogenic factors such AS hyperlipidemia, hypertension and hyperglycemia, oxidized low density lipoprotein (oxLDL) in blood is accumulated under the vascular endothelium, endothelial cells are stimulated to secrete inflammatory factors, mononuclear cells in blood are further recruited to accumulate and adhere under the endothelium, and under the action of cytokines such AS chemokines, the mononuclear cells are differentiated to form macrophages. Macrophages engulf lipid mainly containing oxLDL particles through scavenger receptors such as SR-A, CD on cell membranes in a large amount under intima membrane, lipid droplets are formed in cells and stored, and the macrophages are further converted into foam cells. Excessive uptake of oxLDL AS the main lipid by foam cells causes apoptosis and necrosis, and the lipid is released and accumulated in an endothelial layer to form early characteristic lipid stripes of AS. AS the course of AS progresses, endothelial cells and smooth muscle cells take up oxLDL-based lipids, excessive lipids accumulate in the cells leading to apoptosis and necrosis, and lipids and necrotic cell debris accumulate on the vessel wall, gradually forming the lipid core of atheromatous plaque. Atheromatous plaque causes narrowing of the vessel lumen, obstructing blood flow, and hardening of the vessel wall. And the foam cells secrete a large amount of hydrolase, so that the plaque stability is reduced, the plaque is easy to fall off from the blood vessel wall, and thrombus is formed, and further cardiovascular diseases such as cerebral apoplexy and acute myocardial infarction are caused.

Therefore, the core of AS occurrence is atheromatous plaque formation, macrophage uptake of excessive oxLDL and conversion of the oxLDL into foam cells are important processes of atheromatous plaque formation, and macrophage foaming plays a key role in AS process. Uptake of oxLDL by macrophages is mainly dependent on the expression level of scavenger receptors on their cell membranes, such as CD36 and SR-A. The main methods for clinically surgically treating AS include drug eluting stents and drug coated balloons. The existing scheme for treating AS mainly reduces the blood fat level, and statins, ezetimibe, proprotein convertase subtilisin Kexin type 9 inhibitors and fibrates are used for reducing the level of oxidized low-density lipoprotein cholesterol. However, no specific drug is available to reduce the uptake of oxidized low density lipoprotein cholesterol by cells. If a new treatment strategy can be provided from the perspective of reducing the cellular uptake of oxidized low density lipoprotein, the method has important significance for the prevention and treatment of AS.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an application of miR-204 in diagnosis or treatment of atherosclerosis.

The invention discovers that the expression of microRNA 204 (miR-204 for short) in peripheral blood mononuclear cells of AS patients is reduced compared with that of a normal control group clinically, and miR-204 can effectively reduce the expression of scavenger receptors on macrophage membranes, so that the uptake of oxLDL by macrophages is reduced, and the lipid deposition and foaming of the macrophages are inhibited. Pre-miR-204 is a miR-204 precursor, which is an existing form of miR-204. Pre-miR-204 is cut into two mature bodies with the length of about 20-24 nt by Dicer enzyme digestion, namely miR-204-5p and miR-204-3p. miR-204 has the effect of 'Odongshiki'. On one hand, miR-204-5p inhibits the expression of SR-A in cytoplasm targeting SR-A3' -UTR through a classical pathway. On the other hand, miR-204-3p affects CD36 histone modification in nucleus so as to inhibit transcriptional activation of the histone modification, thereby reducing the expression of CD 36. miR-204 is used AS a novel drug action target, and the detection of peripheral blood miR-204 can be used AS a novel biomarker for diagnosis and prevention of AS diseases.

In order to achieve the purpose, the invention adopts the technical scheme that: provides an application of miR-204 in preparing a medicament for inhibiting lipid deposition and foaming of macrophages.

As a preferred embodiment of the use according to the invention, said inhibition of lipid deposition and foaming of macrophages comprises the prevention or treatment of atherosclerosis.

As a preferred embodiment of the application of the invention, the miR-204 comprises miR-204-5p and miR-204-3p.

The invention aims to overcome the defects of the prior art and determine or discover the microRNA subtype type playing a key role in AS diseases. According to the invention, a large number of experiments show that the expression of pre-miR-204 is reduced in AS diseases. According to the invention, the miR-204 precursor pre-miR-204 and miR-204 mature body (TuD-miR-204-5 p or TuD-miR-204-3 p) function are overexpressed on animals by using an adenovirus method, and the result shows that miR-204 overexpression can obviously inhibit the generation and development of AS, and can close the function of miR-204-5p or miR-204-3p, so that the pathological process can be strengthened or promoted. In addition, the invention also finds that the expression level of pre-miR-204 in Peripheral Blood Mononuclear Cells (PBMCs) of AS patients is reduced clinically.

The invention firstly discovers that the expression of the miR-204 precursor is reduced in the AS process. The expression of pre-miR-204 in PBMCs of a blood sample of a clinical patient is detected by RT-qPCR, and the expression of pre-miR-204 in an AS patient is obviously reduced. In Situ Hybridization (ISH) experiments demonstrated a reduction In pre-miR-204 expression In carotid plaques In AS patients. In animal experiments, with normally fed Apoe ^-/- HFD-fed Apoe compared to mouse ^-/- Expression of pre-miR-204 levels in mouse aorta Dou Bankuai is also reduced.

The invention discovers that in an AS mouse model, the deposition of mouse aorta Dou Zhizhi of AAV-pre-miR-204 injected into tail vein is obviously reduced, the plaque area of aortic sinus is reduced, the necrotic area in plaque is reduced, the plaque stability is increased, and the occurrence and development of AS can be inhibited by over-expressing pre-miR-204. And the function of miR-204-5p or miR-204-3p is sealed, so that the pathological process can be strengthened or promoted.

As a preferred embodiment of the application of the invention, the miR-204-5p is targeted to inhibit SR-A expression so as to inhibit lipid deposition and foaming of macrophages.

As a preferred embodiment of the application of the invention, the miR-204-5p inhibits SR-A through the targeting of a classical seed sequence.

As a preferred embodiment of the application of the invention, the miR-204-3p inhibits the transcription of CD36 so as to inhibit the lipid deposition and foaming of macrophages.

As a preferred embodiment of the application of the invention, the miR-204-3p inhibits the enrichment of H3K27ac and H3K4me3 on a CD36 promoter in a cell nucleus so as to inhibit the transcription of CD 36.

In order to clarify the relevant role of miR-204 in lipid deposition and foam cell formation, by separating Bone Marrow Differentiated Macrophages (BMDMs) of mice, carrying out oil red O staining on the cells and carrying out Uptake and Binding detection on the cells, the transfected miR-204-5p mimics or miR-204-3p mimics are found to obviously inhibit macrophage lipid deposition and foaming. In addition, compared with NC-inhibitor, the transfection of miR-204-5p inhibitor or miR-204-3p inhibitor is found to obviously promote lipid deposition and foaming. The results prove that miR-204-5p and miR-204-3p can inhibit macrophage lipid deposition and foaming. In vitro experiments show that miR-204-5p and miR-204-3p can respectively inhibit the expression of scavenger receptors SR-A and CD 36. Through bioinformatics analysis, an evolutionarily conserved miR-204-5p seed sequence is found in a3 '-untranslated region (3' -UTR) region of SR-A. A luciferase reporter gene experiment is carried out in HEK293T cells, and the results show that miR-204-5p mimics are transfected to reduce the activity of an SR-A3' -UTR reporter gene, and when the miR-204-5p seed region on the SR-A reporter gene is mutated, no influence is caused. The above results demonstrate that miR-204-5p (3 ' -UCCGUAUCCUACUGUUUCCCUU-5 ') inhibits its expression by targeting SR-A3' -UTR. And experiments prove that miR-204-3p does not inhibit the expression of CD36 from a post-transcriptional level, and miR-204-3p is combined with a complementary target site in a CD36 promoter through a specific sequence (3 '-UGCAGGGAAACGGAAGGGUCG-5') in a cell nucleus, so that the enrichment of histones H3K27ac and H3K4me3 on the CD36 promoter is reduced, and the transcription of CD36 is inhibited.

The invention also provides application of the reagent for detecting miR-204 in preparation of a kit for diagnosing atherosclerosis.

As a preferred embodiment of the application of the invention, the miR-204 comprises Pre-miR-204, miR-204-5p and miR-204-3p.

As a preferable embodiment of the application of the invention, the reagent for detecting miR-204 comprises a reagent for detecting the expression level of miR-204.

The invention has the beneficial effects that:

miR-204 has the efficacy of 'Odongshigu', two mature bodies thereof, namely miR-204-5p and miR-204-3p inhibit the expression of a scavenger receptor through different mechanisms in cytoplasm and nucleus respectively, miR-204-5p in the cytoplasm inhibits SR-A expression through a classical seed sequence in a targeted manner, and miR-204-3p inhibits the enrichment of H3K27ac and H3K4me3 on a CD36 promoter in the nucleus so as to inhibit the transcription of CD 36. miR-204 inhibits macrophage foaming and AS through the above approaches in a double-pipe manner.

Drawings

FIG. 1: the expression of miR-204 is reduced in the AS process; wherein, A: collecting peripheral blood of 97 clinical patient samples, wherein the sample grouping condition is 22 normal groups and 75 AS groups, separating PBMCs from the collected peripheral blood, detecting the expression of pre-miR-204 in the PBMCs by using an RT-qPCR technology, and displaying that the expression of the pre-miR-204 in the AS group is lower compared with that of a normal control group; b: ISH staining of pre-miR-204 in carotid arteries of normal and AS-afflicted patients, and Apoe for normal and high-fat feeding ^-/- ISH staining of pre-miR-204 in mice revealed p in AS lesions compared to control groupre-miR-204 expression is reduced.

FIG. 2: the overexpression of pre-miR-204 can inhibit the generation and development of AS; wherein, A: AAV-PCSK9 is injected into tail vein of 8-week-old mice ^DY Starting high-fat feeding to establish an AS mouse model, injecting AAV-LacZ or AAV-pre-miR-204 into tail vein at 12 weeks, continuing high-fat feeding for 20 weeks, taking mouse heart aorta Dou Qiepian and staining ISH of pre-miR-204, wherein the result shows that pre-miR-204 is successfully overexpressed; b: the mouse heart aorta Dou Qiepian after molding in fig. 2A was stained with oil red O to detect lipid deposition. The results show a reduction in lipid deposition in the AAV-pre-miR-204 group compared to the control AAV-LacZ; c: the section obtained after molding in fig. 2A was subjected to hematoxylin-eosin (HE) staining, and the size of the aortic sinus plaque area was examined. The results showed a reduction in aortic sinus plaque area in the AAV-pre-miR-204 group compared to the control AAV-LacZ; d: the section obtained after molding in fig. 2A was subjected to hematoxylin-eosin (HE) staining, and the size of the necrotic area in aorta Dou Bankuai was detected. The results show that the necrosis area of aortic sinus plaque in the AAV-pre-miR-204 group is reduced compared with that of the control AAV-LacZ; e: the section obtained after molding in FIG. 2A was subjected to Masson staining to detect the collagen content in the aorta Dou Bankuai. The results show that compared with the control AAV-LacZ, the AAV-pre-miR-204 group has more collagen content and stronger plaque stability.

FIG. 3: macrophage closes expression of miR-204-3p or miR-204-5p, and generation and development of AS can be promoted; wherein, A: for 8-week-old Apoe ^-/- The mouse is fed with high fat to establish an AS mouse model, AAV-TuD-LacZ, AAV-TuD-miR-204-5p or AAV-TuD-204-3p is injected into tail vein at 12 weeks, high fat feeding is continued for 20 weeks, abdominal cavity macrophages are separated, RNA is extracted to carry out RT-qPCR detection on the expression of miR-204-5p and miR-204-3p, and the result shows that the expression of miR-204-5p and miR-204-3p is obviously reduced. B: the mice molded in fig. 3A were subjected to cardiac aorta Dou Qiepian staining with oil red O to detect lipid deposition. The results showed that lipid deposition was increased in AAV-TuD-204-5p and AAV-TuD-204-3p groups compared with control group AAV-TuD-LacZ. C: the section obtained after the molding in FIG. 3A was subjected to hematoxylin-eosin (HE) staining to detect the area of aortic sinus plaqueOf (c) is used. The results showed that the aortic sinus plaque area was increased in the AAV-TuD-204-5p and AAV-TuD-204-3p groups compared to the control group AAV-TuD-LacZ. D: the section obtained after molding in fig. 3A was subjected to hematoxylin-eosin (HE) staining, and the size of the necrotic area in aorta Dou Bankuai was detected. The results showed that the necrosis area of aortic sinus plaque was increased in the AAV-TuD-204-5p and AAV-TuD-204-3p groups compared to the control group AAV-TuD-LacZ. E: the section obtained after the model fabrication in fig. 3A was subjected to masson staining to detect the collagen content in the aorta Dou Bankuai. The result shows that compared with the control group AAV-TuD-LacZ, the AAV-pre-miR-204 group has less collagen content and the plaque stability is weakened.

FIG. 4: the high-expression miR-204-3p or miR-204-5p can inhibit the lipid deposition and macrophage foaming of macrophages, and the inhibition of the expression of miR-204-3p or miR-204-5p can promote the lipid deposition and macrophage foaming of macrophages; wherein, A: the mouse Bone Marrow Derived Macrophages (BMDMs) are separated, miR-204-5p mimics and miR-204-3p mimics are transfected, after 24 hours, cells are stimulated by oxLDL for 24 hours, oil red O staining is carried out to detect the foaming degree of the cells, and the result shows that the transfected miR-204-5p mimics or miR-204-3p mimics can obviously inhibit lipid deposition and foaming of the macrophages. The miR-204-5p mimics and the miR-204-3p mimics further inhibit the lipid deposition and foaming of macrophages under the synergistic action. B: macrophage (BMDMs) derived from mouse bone marrow are separated, miR-204-5p mimics and miR-204-3p mimics are transfected, cells are stimulated for 2 hours and 4 hours by using Dil marked oxLDL after 24 hours, and the binding and uptake capacity of the cells to the oxLDL is observed under a laser confocal microscope. The result shows that the miR-204-5p mimics or miR-204-3p mimics transfected by the gene can obviously inhibit the lipid deposition and foaming of macrophages. The miR-204-5p mimics and the miR-204-3p mimics further inhibit the lipid deposition and foaming of macrophages under the synergistic action. C: the method comprises the steps of separating macrophages (BMDMs) derived from mouse bone marrow, transfecting miR-204-5p inhibitor and miR-204-3p inhibitor, adding oxLDL to stimulate cells for 24 hours after 24 hours, carrying out oil red O staining to detect the foaming degree of the cells, and displaying that the transfected miR-204-5p inhibitor or miR-204-3p inhibitor can obviously promote the lipid deposition and foaming of the macrophages. The miR-204-5p inhibitor and the miR-204-3p inhibitor further promote the lipid deposition and foaming of macrophages under the synergistic action. D: macrophage (BMDMs) derived from mouse bone marrow are separated, miR-204-5p inhibitor and miR-204-3p inhibitor are transfected, cells are stimulated for 2 hours and 4 hours by using Dil marked oxLDL after 24 hours, and the binding and uptake capacity of the cells to the oxLDL are observed under a laser confocal microscope. The result shows that the miR-204-5p inhibitor or miR-204-3p inhibitor can obviously promote the lipid deposition and foaming of macrophages. The miR-204-5p inhibitor and the miR-204-3p inhibitor further promote the lipid deposition and foaming of macrophages under the synergistic action.

FIG. 5 is a schematic view of: miR-204-5p in cytoplasm inhibits SR-A expression through classical seed sequence targeting so AS to inhibit macrophage foaming and AS; wherein, A: NC-mics and miR-204-5 p-mics are transfected by bone marrow derived mouse mononuclear macrophages (BMDMs), thioglycolate differentiated mouse peritoneal macrophages (TEPMs), human monocyte derived macrophages (HDMDs) and mouse monocyte macrophage leukemia cells (RAW 264.7), and expression of scavenger receptors SR-A and CD36 is detected by Western Blot, and the result shows that compared with NC-mics, miR-204-5 p-mics transfected can obviously inhibit expression of SR-A and does not influence expression of CD 36. B: a specific mechanism that miR-204-5p inhibits the expression of SR-A is explored, and a section of evolutionarily conserved miR-204-5p seed sequence is found in a3 '-untranslated region (3' -UTR) region of SR-A through bioinformatics analysis. C: luciferase reporter gene experiments are developed in HEK293T tool cells, and results show that miR-204-5p mimics transfected reduces SR-A3' -UTR reporter gene activity, and when miR-204-5p on the SR-A reporter gene is combined with a seed region to generate mutation, the effect is avoided. The above results demonstrate that miR-204-5p inhibits the expression of SR-A by targeting SR-A3' -UTR.

FIG. 6: miR-204-3p does not inhibit CD36 expression at post-transcriptional levels; wherein, A: NC-mics and miR-204-3 p-mics are transfected by bone marrow derived mouse mononuclear macrophages (BMDMs), thioglycolate differentiated mouse peritoneal macrophages (TEPMs), human monocyte derived macrophages (HDMDs) and mouse monocyte macrophage leukemia cells (RAW 264.7), and expression of scavenger receptors SR-A and CD36 is detected by Western Blot, and the result shows that compared with NC-mics, miR-204-3 p-mics transfected can obviously inhibit expression of CD36 and does not influence expression of SR-A. B: a specific mechanism of miR-204-3p for inhibiting the expression of CD36 is explored, and a section of miR-204-3p seed sequence which is relatively conserved in evolution is found in a3 '-untranslated region (3' -UTR) region of CD36 through bioinformatics analysis. C: a luciferase reporter gene experiment is developed in a HEK293T tool cell, and the result shows that miR-204-3p mimics transfected have no influence on the activity of a3' -UTR of a CD36 reporter gene, and when a miR-204-3p binding seed region on the CD36 reporter gene is mutated, the activity is also not influenced. D: NC-m, miR-204-3p mimics and mutants thereof (mutation of a seed sequence for combining miR-204-3p mimics and CD 36) are transfected in HDMDs, and the change of the mRNA level of the CD36 is detected. E: NC-m, miR-204-3p mimics and mutants thereof (mutation of a seed sequence for combining miR-204-3p mimics and CD 36) are transfected in HDMDs, and the change of the level of the CD36 protein is detected. F: miR-204-3p mimics were transfected in HMDMs and mRNA expression of CD36 was detected 4 hours later using actinomycin D (5. Mu.g/mL) (transcription inhibition) treatment. As a result, no difference is found in the level change of CD36 mRNA of the transfected NC-mimics and miR-204-3p mimics. The above results demonstrate that miR-204-3p does not inhibit CD36 expression through a classical seed sequence pathway, and does not regulate expression of CD36 through post-transcriptional levels.

FIG. 7 is a schematic view of: miR-204-3p inhibits the enrichment of H3K27ac and H3K4me3 on a CD36 promoter in the cell nucleus so AS to inhibit the transcription of CD36 and further inhibit macrophage foaming and AS; wherein, A: miR-204-3p in situ hybridization and CD68 immunofluorescence co-staining are carried out on the separated BMDMs and HMDMs, and the result shows that miR-204-3p is distributed in the cell nucleus and the cell cytoplasm of macrophages. B: miR-204-3p mimics are transfected in HMDMs, the transcriptional activity of newly synthesized CD36 is measured after 24 hours and 48 hours, and the result shows that miR-204-3p can obviously reduce the transcriptional activity of CD 36. C: potential binding sites of miR-204-3p on a human CD36 promoter and sequences of mutant CD36 promoters (combining site mutation of miR-204-3p and CD36 promoters). D: miR-204-3p mimics are transfected in HEK293T cells, and the luciferase activity of a CD36 promoter or a mutant CD36 promoter is detected after 48 hours. The result shows that miR-204-3p inhibits the activity of a CD36 promoter by binding with the CD36 promoter site. E: miR-204-3p mimics are transfected in HMDMs, a ChIP experiment is carried out by using H3K27ac (enhancer and promoter marker) and H3K4me3 (promoter specificity marker) antibodies after 24 hours, purified and enriched DNA fragments are subjected to sequencing analysis, and the result of UCSC genome-brown traps shows that miR-204-3p can obviously reduce the enrichment of H3K27ac and H3K4me3 on a CD36 promoter. F: miR-204-3p mimics are transfected in HMDMs for 24 hours, an H3K27ac and H3K4me3 antibody is used for performing a ChIP experiment, and a PCR experiment is performed on purified and enriched DNA fragments, so that the miR-204-3p can reduce the enrichment of H3K27ac and H3K4me3 on a CD36 proximal promoter. The above results show that miR-204-3p inhibits the transcription of CD36 by inhibiting the enrichment of H3K27ac and H3K4me3 histones on CD36 promoter in macrophage nucleus.

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

The biological material of the invention is obtained from the following sources:

(1) Human carotid artery AS plaque specimens were provided by the first hospital affiliated with the university of zhongshan.

(2) Normal control and AS patient blood specimens were provided by the second hospital affiliated with the Guangzhou medical university.

(3) HEK293T and RAW264.7 cells were introduced from ATCC in the usa.

(4) C57BL/6 mice were supplied by the laboratory animal center of Zhongshan university, apoe ^-/- The mice were provided by the laboratory animal technology, inc. of Weitonglihua, beijing.

(5)pAAV/D374Y-hPCSK9(PCSK9 ^DY ) The plasmid was supplied by doctor Bentzon.

Example 1 decreased expression of miR-204 in AS Process

(1) Collecting peripheral blood of a clinical patient, separating Peripheral Blood Mononuclear Cells (PBMC) by using a whole blood mononuclear cell separation kit, collecting PBMC to extract RNA, performing reverse transcription to obtain cDNA, and performing fluorescence real-time quantitative PCR to detect the expression of pre-miR-204 in the PBMC. As a result, AS shown in FIG. 1A, the expression of pre-miR-204 was significantly reduced in AS patients.

(2) ISH experiments used a double Digoxigenin (DIG) -labeled Locked Nucleic Acid (LNA) probe to recognize the pre-miR-204 end loop for detection of miR-204 precursor, the sequence of DIG-labeled pre-miR-204 was 5'-GTCCACGAGTCACATGAAGAAAGACTGTAG-3' (SEQ ID NO: 1), paraffin-removed and rehydrated paraffin tissue sections were treated with proteinase K to inactivate endogenous nucleases, and the probes were diluted in hybridization buffer and incubated for 1 hour at 54 ℃. After in situ hybridization, observation is carried out under a microscope. The results are shown in FIG. 1B, with a decrease in pre-miR-204 expression in AS patients and mouse carotid plaques.

Example 2 miR-204 inhibits AS formation

(1) AAV-pre-miR-204, AAV-TuD-miR-204-5p and AAV-TuD-miR-204-3p are prepared.

The entire sequence of the miR-204 precursor (pre-miR-204, SEQ ID NO. The plasmids are respectively co-transfected into HEK293T cells together with pAAV2/8 reverse plasmid and pAAV helper plasmid carrying AAV rep/cap gene by PEI liposome transfection method to generate AAV8 adenovirus (AAV-pre-miR-204, AAV-TuD-miR-204-5p and AAV-TuD-miR-204-3 p), and AAV-LacZ is used as negative control. Viral titers were measured by PCR with vector-specific primers.

(2) Preparation of AS mouse model

AAV-PCSK9 ^DY And (5) establishing an induced mouse AS model. AAV-PCSK9 ^DY Can target and recognize the LDL receptor of the liver, degrade the LDL-R in lysosome and have similar action to the LDL-R ^-/- Mice induced AS. For the construction of AAV-PCSK9 ^DY pAAV/D374Y-hPCSK9 driven by ApoEHCR-hAAT promoter (PCSK 9) ^DY ) The plasmids respectively carry AAV rep&Co-transfection of the pAAV2/8 antiplasmid and pAAV helper plasmids of the cap genes into HEK293T cells to generate AAV8 adenoviruses (AAV-PCSK 9) ^DY ) AAV-LacZ was used as a negative control. By using vector-specific primersThe virus titer was measured by PCR. We injected AAV-PCSK9 to tail vein of 8 week old male mice ^DY (1×10 ¹¹ vg/mouse), mice were given High Fat Diet (HFD) (40% fat, 11.4% sucrose, 1.25% cholesterol), and fed for 12 weeks to induce an AS disease model.

Apoe ^-/- And (5) establishing an induced mouse AS model. The invention selects the Apoe with SPF grade of 8 weeks old ^-/- Male mice were given High Fat Diet (HFD) (40% fat, 11.4% sucrose, 1.25% cholesterol) and fed for 12 weeks to induce an AS disease model. The environmental temperature of all experimental mice is 20-25 ℃, the relative humidity is 50-60%, and the 12-hour illumination period simulates circadian rhythm. All animals were housed in the SPF animal laboratory, the animal laboratory center of university of zhongshan, and the experiment was approved by the animal ethics committee of university of zhongshan.

(3) Results of the experiment

The results are shown in FIG. 2, by comparing AAV-PCSK9 ^DY AAV-pre-miR-204 is injected into the induced AS mouse through tail vein, the lipid deposition of the mouse is obviously reduced, the plaque area of aortic sinus is reduced, the necrotic area in the plaque is reduced, and the stability of the aorta Dou Bankuai is improved, which shows that the occurrence and development of AS can be inhibited by over-expressing pre-miR-204.

As shown in fig. 3, by applying Apoe ^-/- The pathological process can be strengthened or promoted by injecting AAV-TuD-miR-204-5p and AAV-TuD-miR-204-3p into mouse tail vein to seal the expression of macrophage miR-204-5p or miR-204-3p. RT-qPCR results show that the expression of miR-204-5p or miR-204-3p is remarkably inhibited. And the blocking of the expression of macrophage miR-204-5p or miR-204-3p can strengthen or promote the pathological process.

Example 3 miR-204 can inhibit macrophage lipid deposition and foaming

To elucidate the relevant role of miR-204 in lipid deposition and foam cell formation, cells were stained for oil red O and the uplike and Binding assay for Dil-labeled oxLDL by isolating mouse BMDMs (mouse bone marrow-derived macrophages).

The result is shown in FIG. 4, and the miR-204-5p mimics or miR-204-3p mimics transfected by the method can obviously inhibit lipid deposition and foaming. Compared with NC-inhibitor, miR-204-5p inhibitor or miR-204-3p inhibitor transfected by the method can obviously promote lipid deposition and foaming. The results prove that the overexpression of the miR-204-5p and the miR-204-3p can inhibit macrophage lipid deposition and foaming.

Wherein, the miR-204-3p mimics and the miR-204-5p mimics are synthesized and provided by Ji Ma of Suzhou, and have the following sequences:

example 4 miR-204-5p inhibits its expression by targeting the scavenger receptor SR-A3' -UTR

miR-204-5p mimics are transfected in various macrophages in vitro, and cell proteins are extracted for carrying out Western blotting to find that miR-204-5p can obviously reduce the expression of the scavenger receptor SR-A, and has no influence on the scavenger receptor CD 36. In view of the fact that miRNA can inhibit the expression of a target gene by targeting a3' -untranslated region (3 ' -UTR) of target gene mRNA, a section of miR-204-5p seed sequence which is evolutionarily conserved is found in the 3' -UTR region of SR-A through bioinformatics analysis.

Luciferase reporter experiments were performed in HEK293T tool cells: inserting a3' -UTR sequence of SR-A mRNA into a reporter gene vector, co-transferring the mRNA into miR-204-5p mimics, and detecting the luciferase activity after 48 hours. The result is shown in FIG. 5C, miR-204-5p mimics are transfected to reduce the activity of the SR-A3' -UTR reporter gene, and when the miR-204-5p seed region on the SR-A reporter gene is mutated, the influence is avoided. The above results demonstrate that miR-204-5p (3 ' -UCCGUAUCCUACUGUUUCCCUU-5', SEQ ID NO: 10) inhibits its expression by targeting SR-A3' -UTR.

Example 5 miR-204-3p inhibits the transcription of the scavenger receptor CD36 in the nucleus

miR-204-3p mimics are transfected in various macrophages in vitro, and cell proteins are extracted for carrying out Western blotting to find that miR-204-3p can obviously reduce the expression of the scavenger receptor CD36 without influencing the scavenger receptor SR-A.

Next, the mechanism of miR-204-3p for inhibiting CD36 expression is explored, and a section of miR-204-3p seed sequence which is relatively conserved in evolution is found in the 3' -UTR region of CD 36. Therefore, luciferase reporter experiments were developed in HEK293T tool cells: inserting a3' -UTR sequence of CD36 mRNA into a reporter gene vector, co-transferring into miR-204-3p mimics, and detecting luciferase activity after 48 hours. As shown in FIG. 6C, the miR-204-3p mimics transfected by the gene has no influence on the activity of the CD36 3' -UTR reporter gene, but has no influence when the miR-204-3p seed region on the CD36 reporter gene is mutated. In addition, human monocyte-derived macrophages (HMDMs) are isolated, miR-204-3p mimics and miR-204-3p mimics mut (a mutant of a predicted miR-204-3p and CD36 mRNA 3' -UTR binding sequence) are transfected, and then the CD36 protein and mRNA levels are detected, so that the result shows that miR-204-3p can remarkably reduce the CD36 protein and mRNA levels, and the CD36 protein and mRNA levels are still reduced after the binding sequence is mutated. This indicates that miR-204-3p does not inhibit the expression of CD36 by this seed sequence.

Isolated HMDMs were transfected with miR-204-3p mimics, and incubated with actinomycin D (ActD, 5. Mu.g/mL) 4 hours later to inhibit mRNA synthesis and interfere with the transcription process of the cells. The results are shown in FIG. 6, and miR-204-3p does not change the half-life of CD36 mRNA. This indicates that miR-204-3p does not inhibit the expression of CD36 from the post-transcriptional level.

The results of our study found that miR-204-3p affects the transcription of CD36 in the nucleus. First, miR-204-3p in-situ hybridization and CD68 immunofluorescence co-staining are carried out on separated BMDMs and HMDMs, and the result shows that miR-204-3p is distributed in the nucleus and the cytoplasm of macrophages. miR-204-3p can obviously reduce the transcriptional activity of CD 36. Through RNAhybrid sequence prediction analysis, a conserved miR-204-3p potential binding site is located 100bp upstream of a CD36 promoter transcription initiation site. miR-204-3p mimics are then transfected in HEK293T cells, and luciferase activity of the CD36 promoter or mutant CD36 promoter is detected 48 hours later. The results are shown in FIG. 7, and miR-204-3p inhibits CD36 promoter activity by binding to CD36 promoter site. miR-204-3p mimics are transfected in HMDMs, a ChIP experiment is carried out by using H3K27ac and H3K4me3 antibodies after 24 hours, purified and enriched DNA fragments are subjected to sequencing analysis, and the result of UCSC genome-brown traps shows that miR-204-3p can obviously reduce the peak value of H3K27ac and H3K4me3 on a CD36 promoter. miR-204-3p mimics are transfected in HMDMs, a ChIP experiment is carried out by using H3K27ac and H3K4me3 antibodies after 24 hours, PCR experiment is carried out on purified and enriched DNA fragments, and the fact that miR-204-3p can reduce the enrichment of H3K27ac and H3K4me3 on a CD36 proximal promoter is clear. The above results show that miR-204-3p is combined with a complementary target site in a CD36 promoter through a specific sequence (3 '-aUGCAGGGAAACGGAAGGGUCG-5', SEQ ID NO: 11) in a macrophage nucleus, so that the enrichment of histones H3K27ac and H3K4me3 on the CD36 promoter is reduced, and the CD36 transcription is inhibited.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Zhongshan university

<120> application of miR-204 in diagnosis or treatment of atherosclerosis

<130> 2022.01.05

<160> 11

<170> PatentIn version 3.3

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Claims

Application of miR-204-5p mimics or miR-204-3p mimics in preparation of medicines for treating atherosclerosis;

the sequence of the sense chain of the miR-204-5p mimics is shown as SEQ ID NO:4 is shown in the specification;

the sequence of the antisense chain of the miR-204-5p mimics is shown in SEQ ID NO:5 is shown in the specification;

the sequence of the sense chain of the miR-204-3p mimics is shown as SEQ ID NO:6 is shown in the specification;

the sequence of the antisense chain of the miR-204-3p mimics is shown in SEQ ID NO: shown at 7.
2. The use of claim 1, wherein the miR-204-5p mimics are targeted to inhibit SR-a expression and thus inhibit lipid deposition and foaming of macrophages.
3. The use according to claim 1, wherein the miR-204-3p mimics inhibits transcription of CD36 and thus inhibits lipid deposition and foaming of macrophages.
4. The use of claim 1, wherein the miR-204-3p mimics inhibit the enrichment of H3K27ac and H3K4me3 on a CD36 promoter in the cell nucleus so as to inhibit the transcription of CD 36.