Study On The Regulation Mechanism Of MiR-149-3p In Atherosclerosis

BackgroundAtherosclerosis is the main cause of cardiovascular and cerebrovascular diseases such as coronary heart disease,myocardial infarction and stroke,and the disorder of lipid metabolism is the basis of atherosclerosis.Micro RNA（miRNA）is a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of about 22 nucleotides,which are involved in the regulation of post-transcriptional gene expression.Our previous studies found that the expression of miR-149-3p was significantly increased in mouse models and clinical samples with abnormal lipid metabolism.Therefore,miR-149-3p can be used as a clinical diagnostic marker and may regulate lipid metabolism disorders.In order to explore the molecular mechanism of miR-149-3p in abnormal lipid metabolism and atherosclerosis,we established the LDLR^-/-miR-149-3p^-/-double gene knockout mouse model in LDLR^-/-mice with lipid metabolism disorder.This study may provide experimental basis for accurate diagnosis and treatment of cardiovascular and cerebrovascular diseases.ObjectiveThe double-gene knockout mouse model was established to explore the role and molecular mechanism of miR-149-3p in lipid metabolism disorders and atherosclerosis.Method1.The double gene knockout mouse model of LDLR^-/-miR-149-3p^-/-was establishedBased on the background of LDLR^-/-mice with lipid metabolism disorder,the model of LDLR^-/- miR-149-3p^-/-double gene knockout mice was established,and then backcrossed with LDLR^-/-mice for more than 3 generations to establish LDLR^-/-miR-149-3p^-/-murine-mice with clear genetic background.Male mice of 8-10 weeks LDLR^-/-miR-149-3p^+/+and LDLR^-/-miR-149-3p^-/-were selected and fed in SPF barrier facilities on Western diet for 0-8 weeks,with 6-8 mice in each group.Fasting without water for 6 h,anesthesia with tribromo-ethanol,blood from the eye canthus vein,plasma was treated with heparin sodium anticoagulation.The liver,heart and intestinal tissues of mice were frozen in liquid nitrogen and all samples were preserved at-80℃.This study was repeated at least 3 times independently.2.miR-149-3p regulates lipid and bile acid metabolism in miceDuring 0-8 weeks of Western diet,plasma,liver,bile and feces were collected regularly to detect the levels of triglyceride,cholesterol and bile acid.The mouse plasma was diluted 4-fold with PBS,and plasma lipoproteins were separated by fast protein liquid chromatography（FPLC）.The lipoproteins were eluted at the rate of 0.5 m L/min by Superose6 HR10/30 column.The Amplex^TMRed Cholesterol Ultra Sensitive Kit was used to detect the levels of different components of lipoprotein cholesterol isolated by FPLC.3.miR-149-3p regulates atherosclerosis and fatty liver in miceAfter 8 weeks of Western diet,the hearts of mice were isolated and perfused with PBS,the aortic arch was peeled off,and the size of atherosclerotic plaque of aortic arch was observed under stereoscope.Liver and heart tissues of mice were rapidly fixed,frozen sections were performed,and the changes of atherosclerotic plaque area in aortic sinus and fatty liver of mice were observed by oil red O staining.The expression of CD36 in mouse heart plaque and liver tissue was detected by immunohistochemical staining.4.miR-149-3p regulates genes related to lipid metabolism and vascular inflammation in mouse liverTranscriptome sequencing of liver mRNA was performed using Illumina Hiseq high-throughput sequencing technology,and GO and KEGG functional annotation and enrichment analysis were performed to screen out differentially expressed genes and pathways related to lipid metabolism.qRT-PCR and Western blot were used to detect the differences in the expression levels of genes related to lipid metabolism and vascular inflammation in liver tissues.5.miR-149-3p regulates intestinal flora structure and cholesterol absorption in miceThe fecal samples of mice in the two groups were sequenced by 16S rDNA.OTU clustering analysis,Alpha diversity analysis,Beta diversity analysis,phylum level and genus level difference analysis were used to study the abundance,diversity and species composition of intestinal microflora in the two groups.The mice of the two groups after Western diet were fasted for 12 hours and fed with NBD-cholesterol,and blood samples were collected 4 h later to detect serum fluorescence value.The intestinal tissues were divided into 5 sections and frozen sections were taken respectively,and the absorption and distribution of exogenous cholesterol were observed under fluorescence microscope.6.Identification of target genes of miR-149-3pmiR-149-3p Ago,miR-149-3p ANT and control groups were transfected into HepG2 cells,and mRNA was extracted from the cells.qRT-PCR and Western blot were used to detect the expression levels of lipid metabolism-related genes in HepG2 cells.In order to further verify whether ABCG5 and ABCG8are the target genes of miR-149-3p,dual luciferase reporter gene vectors were constructed by predicting the binding site of miRNA and 3’-UTR in Targetscan,miRWalk and other databases.After plasmid transformation,amplification,extraction and purification,high purity recombinant double luciferase vectors of ABCG5 and ABCG8 were obtained,and co-transfected with miR-149-3p Ago,miR-149-3p ANT and their controls to detect the activity of recombinant double luciferase vectors.Result1.miR-149-3p regulates lipid and bile acid metabolism in miceCompared with the control group,the plasma total cholesterol and triglyceride levels of LDLR^-/- miR-149-3p^-/-mice were significantly decreased,and the total bile acid levels were significantly decreased at the 8th week.FPLC analysis showed that compared with the control group,the plasma VLDL-C and LDL-C levels of LDLR^-/-miR-149-3p^-/-mice were significantly decreased,while the levels of HDL-C were significantly increased.The level of total cholesterol in gallbladder bile of LDLR^-/-miR-149-3p^-/-mice decreased significantly,but the level of triglyceride did not change significantly,while the level of total bile acid increased significantly.The levels of total cholesterol（TC）and triglyceride（TG）in feces of LDLR^-/-miR-149-3p^-/-mice decreased significantly,while the levels of total bile acid increased.2.miR-149-3p regulates atherosclerosis and fatty liver in miceThe plaque of aortic arch in LDLR^-/-miR-149-3p^-/-mice decreased significantly.Oil red O staining showed that the area of plaque in cardiac sinus and the content of lipid droplets in hepatocytes decreased significantly.The results of immunohistochemical staining showed that the number of CD36positive cells in the heart and liver of LDLR^-/-miR-149-3p^-/-mice decreased significantly.3.miR-149-3p regulates lipid metabolism and inflammation-related genes in mouse liver,blood vessel and intestineA total of 275 differentially expressed genes were screened by gene sequencing in the liver transcriptional group,including 110 up-regulated genes and 165 down-regulated genes.The results of qRT-PCR and Western blot showed that the expression levels of ABCG5,ABCG8,APOA1,CYP7A1,CYP7B1 and BSEP in the liver of LDLR^-/-miR-149-3p^-/-mice were significantly increased,while the expression levels of SREBF2,LXRα,APOB,ABCG1,NTCP,CYP8B1,SHP,APOC3,ACACA,E2F1 and SREBP-1c were significantly decreased.The expression levels of vascular inflammation related genes CD209,MCP-1,CD68,CD86,IFNγ,CD32,TGFβ,VCAM1 and F4/80 decreased significantly.The expression of cholesterol absorption related genes NPC1L1 and NUMB decreased significantly,while the expression levels of ABCG5,ABCG8,SR-B1 and LXRαincreased significantly in intestinal tissue of mice.4.miR-149-3p regulates intestinal flora structure and cholesterol absorption in miceThe results of intestinal flora sequencing showed that the abundance of Lactobacillus decreased and the abundance of Bifidobacterium and Rosella increased in LDLR^-/-miR-149-3p^-/-mice.Intestinal flora sequencing results showed that the abundance of Lactobacillus in LDLR^-/-miR-149-3p^-/-mice decreased,while the abundance of Bifidobacterium and Rosella increased.Alpha diversity index showed an upward trend as a whole,and the results of Beta diversity analysis showed that there were significant differences in intestinal flora between the two groups.According to the prediction of differential metabolic pathways in KEGG database,it was found that there were significant differences in 56 secondary metabolic pathways among the 6 major metabolic pathways.The absorption of cholesterol in the intestine of LDLR-/-miR-149-3p mice was significantly decreased.5.Identification of target genes of miR-149-3pIn HepG2 cells,miR-149-3p gene was overexpressed,and the expression levels of ABCG5 and ABCG8 genes were significantly decreased.When miR-149-3p gene was knocked down,the expression levels of ABCG5 and ABCG8 genes were significantly increased.Double luciferase reporter gene results showed that overexpression of miR-149-3p gene could significantly reduce the activity of double luciferase vector ABCG5 and ABCG8,and knockdown of miR-149-3p gene could significantly improve the activity of double luciferase vector in both groups.It was proved that ABCG5 and ABCG8 were the direct target genes of miR-149-3p.Conclusion1.miR-149-3p knockout significantly decreased the levels of plasma VLDL-C and LDL-C and increased the level of HDL-C in mice.2.miR-149-3p knockout significantly reduced the area of atherosclerotic plaque and the content of fat in liver cells in mice.3.miR-149-3p regulates the diversity and richness of intestinal flora in mice,and inhibits the absorption of intestinal exogenous cholesterol.4.ABCG5 and ABCG8 were the potential target genes of miR-149-3p.