The Study On The Effect And Mechanism Of MiR-31 Inhibited The Apoptosis Of Endothelial Cells Induced By Angiotensin-2

Purpose:Hypertension,one of the most common cardiovasc?Lar disease,affects as many as 1 billion people worldwide.Despite the continuous improvement of medical development,currenttly the incidence of hypertension is high,and the rate of disease awareness and treatment and rate of blood pressure control are still low in our country.Endothelial cells(ECs)are the most directly affected enveloped cells after an increase in blood pressure.Dysfunction of ECs can lead to the release of inflammatory mediators and procoagulant factors,the decline of the migration capability,and dysfunction of vasodilation,which ultimately leads to serious complications of heart,brain,kidneys,eyes,and blood vessels.At present,ECs,renin-angiotensin-aldosterone system(RAAS)and other factors are considered to be associated with the pathogenesis of hypertension.Angiotension ?(Ang ?)as the most important vasoactive substance in RAAS not only can cause hemodynamic abnormalities leading to hypertension,but also play an important role in endothelial function.Therefore,it is particularly important to study the mechanism of endothelial cell damage caused by hypertension.MicroRNAs(miRNAs),as an endogenous non-coding RNA,bind to the 3 'untranslated region(UTR)of targeted mRNAs,rendering their translation blocked or causing degradation of the targeted mRNAs.In recent years,a large number of literatures have reported that miRNA can regulate cardiovascular diseases by affecting the biological processes of cell apoptosis,proliferation,metabolism,and differentiation.Some studies have found that miR-31 palys a regulatory role in myocardial fibrosis,vascular smooth muscle cells(VSMCs)phenotype transformation,and myocardial ischemia/reperfusion injury.However,the role and mechanism of miR-31 in endothelial dysfunction in hypertensive disease is not clear.In this study,we aim to investigate the effect and mechanism of miR-31 on the function of endothelial cells in the pathogenesis of hypertension.Methods:1.Human umbilical vein endothelial cells were isolated by enzyme digestion;HUVECs were induced by Ang ?(1?mol/L)to establish endothelial cell injury model.The biological function of endothelial cells was tested by Flow cytometry analysis,western Blot and CCK-8 assay.The expression of miRNA was detected,by quantitative real-time polymerase chain reaction(qRT-PCR).2.HUVECs were transfected with miR-31 mimic/inhibitor respectively and the transfection efficiency was confirmed by qRT-PCR.The biological functions of HUVEcs over-expressing and interfering miR-31 were detected,including plate scratch test,CCK-8 assay and flow cytometry analysis.3.Bioinformatics prediction software searches for potential target genes of miR-31 and analyzes binding sites of miR-31 and 3'UTR of potential target genes;The constructed firefly luciferase expression vector was co-transfected into 293 T cells and the regulatory relationship between miR-31 and RASA1 was verified by luciferase reporter assay and Western Blot;the mechanism of miR-31 and target genes in HUVECs injury induced by Ang ? was clarified through rescue experiments.Reults:1.CCK8 assay showed that a significant decrease in endothelial cells viability(P <0.01),flow cytometry suggested that apoptosis increased After HUVECs were induced by Ang ?(P <0.01),western blot showed that the expression of Caspase-3 was up-regulated and expression of Bcl-2was down-regulated.In addition,there was no significant difference in the expression of miR-126,miR-200 and miR-221(P>0.05),while the expression of miR-155 was up-regulated(P <0.01),and the expression of miR-31,miR-384,and miR-590-5p are down-regulated(P <0.01).The expression of miR-31 decreased to some extent respectively at different time(P <0.01),especially HUVECs were induced by Ang ? after 24 h.2.CCK8 assay showed that overexpression of miR-31 can increase the viability of HUVECs(P <0.01)and downregulation of miR-31 can decrease the viability of HUVECs(P <0.01).At the same time,flow cytometry showed that the increased miR-31 expression significantly reduce the apoptosis of HUVECs(P <0.01),and the decreased miR-31 expression significantly enhance the apoptosis of HUVECs(P <0.01).Scratch wound healing assay demonstrated that the increased miR-31 expression significantly up-regulated the migration capability of HUVECs,while the decreased miR-31 expression significantly down-regulated the migration capability of HUVECs3.The bioinformatics analysis showed that the potential binding site of miR-31 exists on the 3'UTR of RASA1 gene.The Dual-luciferase assay confirmed that over-expression of miR-31 significantly reduced the luciferase activity of the 3 'untranslated region of RASA1(P <0.01),Western blot show that over-expression of miR-31 can significantly inhibit expression of RASA1 protein(P <0.01).The rescue experiments confirm that overexpression of miR-31 protects HUVECs against apoptosis induced by Ang ? by inhibiting RASA1 expression.Conclusion:1.The expression of miR-31 in endothelial cells induced by Ang ? was down-regulated,suggesting miR-31 is involved in the regulation of Ang ?-induced endothelial cell injury.2.Over-expression of miR-31 inhibits Ang ?-induced apoptosis in endothelial cells and promotes endothelial cell migration.3.The protective effect of miR-31 on Ang ?-induced apoptosis in endothelial cells is achieved by inhibiting RASA1 expression.