MicroRNA-377 Regulates Triglyceride Metabolism And Atherosclerosis And Its Mechanism

[Background and Objective]:Atherosclerosis(AS)is the common pathological basis of cardiovascular and cerebrovascular diseases.Lipid metabolism disorder,such as the aberrant elevation of triglyceride level,is an independent risk factor for AS.Increasing lines of evidence suggest that micro RNAs(mi RNAs)have emerged as critical regulators in lipid metabolism and atherosclerosis.Our colleagues have previously shown that mi R-186,mi R-134,and mi R-467 b induce lipid accumulation and activate inflammatory response in macrophages.As a polyphonic mi RNA,mi R-377 is known to be involved in inflammation,oxidative stress and angiogenesis in ischemic hearts.In addition,mi R-377 has been demonstrated to regulate the cardiac regenerative ability.It was reported that mi R-377 levels were closely associated with lipid levels in vivo,suggesting that circulating mi R-377 might play an important role in lipid metabolism.To date,there is no report about the role of mi R-377 in the development of AS.DNA methyltransferase 1(DNMT1)is one of the members in DNMTs family.As one of the most important DNA methyltransferases,DNMT1 is to maintain the methylated state after DNA replication and guarantee this methylation status to be passed on to the progeny cells.It was reported that overexpression of DNMT1 in macrophages induced pro-inflammatory cytokines production and atherosclerosis development.Moreover,DNMT1 is associated with elevated triglyceride levels.However,the substrate of DNMT1 involved in triglyceride metabolism has not yet been fully elucidated.Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1(GPIHBP1),as a GPI-anchored protein of capillary endothelial cells,it is to combine with lipoprotein lipase(LPL)stably in the sub-endothelial space,then transport LPL to the capillary lumen.After GPIHBP1 binding to LPL,triglyceride-rich lipoproteins(TRLs)(such e.g.chylomicrons and very low-density lipoprotein)migrated to the surface of capillary cells,which promotes LPL hydrolysis of triglycerides.Taken together,the aim of this study was to explore the molecular mechanism of mi R-377 regulating triglyceride metabolism and AS.Our findings have indicated that mi R-377 decreases the DNA methylation levels of GPIHBP1 gene promoter through silencing DNMT1,then increases the binding of LPL to GPIHBP1 on the surface of endothelial cells and decreases the plasma triglyceride levels,inhibits the development of AS in apo E-/-mice.The result suggests that mi R-377 may act as a novel pharmacologic target for atherosclerosis.[Methods]:The clinical data of patients with coronary atherosclerotic heart disease(CHD)were collected and arranged,we divided it into two groups,including CHD with hyperlipidemia and CHD without hyperlipidemia.The m RNA levels of lipid metabolism-associated micro RNAs(mi RNAs)in the plasma was detected by RT-q PCR.The correlation between low expression of mi R-377 and high triglyceride level of plasm was analyzed.Using micro RNA.org-Targets and Expression,mi Rna Viewer and RNA hybrid,we observed a predicted binding site and free energy score for mi R-377 in the 3’ untranslated region(3’-UTR)of DNMT1.Moreover,we validated the combination between mi R-377 and DNMT1,mi R-377 and GPIHBP1,mi R-377 and LPL through luciferase reporter gene detection.We transfected mi R-377 mimic and mi R-377 inhibitor at different concentrations(0,10,20,40,80 n M)or different time(0,6,12,24,48 h)into human umbilical vein endothelial cells(HUVECs).Then,the effects of mi R-377 on DNMT1 activity in endothelial cells were tested by DNA methyltransferase activity colorimetric assay kit.The m RNA and protein levels of DNMT1 were detected by RT-q PCR and Western blot analysis,respectively.Using bioinformatic analyses and NCBI database,cpgislands on the website line,we analyzed Cp G islands in GPIHBP1 gene promoter.Using adenovirus vector transfected DNMT1,sh DNMT1 or treated with DNMT1 inhibitor 5-nitrogen heterozygous-2 ’deoxycytidine(5-Aza-Cd R)in cells.The DNA methylation levels of GPIHBP1 promoter were detected by bisulfite sequencing.The m RNA and protein levels were tested by RT-q PCR and Western blot,respectively.After transfected into cells with mi R-377 mimic,DNMT1,mi R-377 mimic + DNMT1,or mi R-377 mimic + sh DNMT1,we used bisulfite sequencing to determine the DNA methylation levels of GPIHBP1 promoter,detected GPIHBP1 m RNA and protein levels by RT-q PCR and Western blot.The GPIHBP1 and LPL binding assay to ascertain the binding of LPL to GPIHBP1 on the surface of endothelial cells.Male eight-week-old Apo E-/-mice were randomly divided into 4 groups: mi R-377 scrambled agomir negative control group(AG-NC),mi R-377 agomir group(AG),mi R-377 scrambled antagomir negative control group(AN-NC),and mi R-377 antagomir group(AN).Mice were fed high-fat/high-cholesterol diet and received a tail vein injection of mi R-377 agomir,mi R-377 antagomir or one of the corresponding controls at a dose of 80mg/kg wt every 2 weeks.After 12 weeks on the high-fat/high-cholesterol diet,mice aorta endothelial cells(MAECs)were extracted,the m RNA levels of DNMT1 and GPIBHP1 were dected by RT-q PCR,the protein levels of DNMT1 and GPIHBP1 were dected by Western blot.DNA methyltransferase activity colorimetric assay kit examined DNMT1 activity.The GPIHBP1 and LPL binding assay to ascertain the binding of LPL to GPIHBP1 on the surface of MAECs.Automatic biochemical analyzer detected plasma lipid levels in mice.The aortic arch and its three major branches(left subclavian artery,left common carotid artery and truncus brachiocephalicus artery)were isolated from each group of mice.Stereoscopic microscopic observed the AS plaque area in the aortic arch.The aortic arch was split longitudinally to the branches of the common iliac artery,and the lipid accumulation in the aortic wall was observed by oil red O staining.The aortic sinus sections were sectioned by rapidly frozen sections technique.The plaque area,lipid accumulation and collagen content in aortic sinus were detected by HE,Oil red O,and Masson’s trichrome staining,respectively.[Results]:According to the clinical data of patients with coronary atherosclerotic heart disease and RT-q PCR,mi R-377 m RNA level was reduced in plasma with hypertriglyceridemia.It suggests that mi R-377 may have biological effects of regulating triglyceride metabolism.Using micro RNA.org-Targets and Expression,we observed a predicted binding site for mi R-377 in the 3’ untranslated region(3’-UTR)of DNMT1.The sequence for mi R-377 binding is evolutionarily conserved among various animal species.Several target gene prediction algorithms suggested that mi R-377 had highly homologous with a sequence in the 3’-UTR of human DNMT1 m RNA.Moreover,the conservative combining site of mi R-377 and DNMT1 is highly conserved among different species.In addition,the free energy scores for the hybridization between mi R-377 and DNMT1 is low in human.These bioinformatics prediction results suggest that mi R-377 is a potential important regulator of DNMT1.We used Target Scan found that mi R-377 has no binding site with GPIHBP1 or LPL 3’-UTR.On these bases,we constructed a wildtype and a mutant DNMT1 3’-UTR luciferase reporter vector with a mi R-377 homologous sequence.MiR-377 mimic decreased the relative luciferase activity of the wildtype reporter but had no effect on the mutant one when compared with the control group in HEK 293 T cells.Furthermore,mi R-377 had no effect on the luciferase activity of GPIHBP1 and LPL wildtype.In addition,mi R-377 mimic inhibited DNMT1 expression both in concentration and time-dependent manners.But,cells treated with mi R-377 inhibitor can reverse these effects.Overexpression DNMT1 significantly increased the methylation levels of GPIHBP1 promoter,and decreased GPIHBP1 expression.However,cells transfected with sh DNMT1 or treated with 5-Aza-Cd R reversed these results.Cells transfected with mi R-377 mimic obviously decreased the DNA methylation levels of GPIHBP1 promoter,then upregulated GPIHBP1 expression and increased the binding of LPL to GPIHBP1 on the surface of endothelial cells.The results of mi R-377 inhibitor treatment group were opposite.MiR-377 mimic and DNMT1 co-transfect group significantly inhibited DNMT1-induced GPIHBP1 promoter DNA methylation,then upregulated GPIHBP1 expression and increased the binding of LPL to GPIHBP1 on the surface of endothelial cells.Also,mi R-377 inhibitor and sh DNMT1 co-treatment group had an opposite effect.In MAECs,mi R-377 agomir decreased DNMT1 expression and activity,upregulated GPIHBP1 expression and increased the binding of LPL to GPIHPB1 on the surface of MAECs,reduced plasma lipid levels.MiR-377 antagomir had an opposite effect.In apo E-/-mice,mi R-377 agomir reduced atherosclerotic plaque area,inhibited the lipid deposition in the atherosclerotic plaque of aorta and decreased aortic sinus plaque area and lipid deposition,but increased the collagen content in aortic sinus.MiR-377 antagomir had an opposite effect.[Conclusion]:MicroRNA-377 inhibits DNMT1 expression and activity by specifically combining with its 3’-UTR,suppresses DNA methylation in GPIHBP1 promoter,upregulates GPIHBP1 expression,thereby increases the binding of LPL to GPIHBP1 on the surface of endothelial cells.Besides,mi R-377 reduces plasma triglyceride levels and suppresses the formation of atherosclerotic plaque in Apo E-KO mice.