Objective To investigate the regulatory role of microRNA-363-5p(miR-363-5p)in myocardial hypertrophy by targeting thrombospondin-3(THBS3).Methods An in vitro model of myocardial hypertrophy was replicated by treating human myocardial cells(AC 16)with angiotensin Ⅱ(AngⅡ).The cytoskeleton was observed with the phalloidin staining,and Western blotting was used to evaluate the protein expression levels of embryonic genes in the cell model of myocardial hypertrophy to confirm the successful model establishment.Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to detect the expression level of miR-363-5p in the cell model of myocardial hypertrophy.Western blotting was applied to assess the changes in hypertrophy-related phenotypes in hypertrophic myocardial cells after transfection with miR-363-5p mimics and miR-363-5p inhibitor.Dual-luciferase reporter assay was conducted to validate the binding of miR-363-5p to the 3'-UTR of THBS3.Rescue experiments with overexpression of both THBS3 and miR-363-5p were designed to evaluate whether THBS3 mediated the regulatory role of miR-363-5p in myocardial hypertrophy.Results The area of cells in the AngⅡgroup was larger than that in the control group(P＜0.05).The expressions of ANP,BNP,β-MHC and miR-363-5p in the AngⅡ group were higher than those in the control group(P＜0.05).The relative expression of miR-363-5p in the miR-363-5p mimics group was higher than that in the mimics-NC group(P＜0.05),while that in the miR-363-5p inhibitor was lower compared with the inhibitor-NC(P＜0.05).The relative expressions of ANP,BNP and β-MHC in the miR-363-5p mimics group were lower than those in the mimics-NC group(P＜0.05),while those in the miR-363-5p inhibitor were higher compared with the inhibitor-NC group(P＜0.05).The area of cells in the miR-363-5p mimics group was smaller than that in the mimics-NC(P＜0.05),while that in the miR-363-5p inhibitor group was larger compared with the inhibitor-NC group(P＜0.05).The luciferase activity of THBS3-WT in the miR-363-5p mimics+THBS3-WT group was lower than in the mimics-NC+THBS3-WT group(P＜0.05).There was no difference in the luciferase activity of THBS3-MUT between the mimics-NC+THBS3-MUT group and the miR-363-5p mimics+THBS3-MUT group(P＞0.05).The relative mRNA and protein expressions of THBS3 in the miR-363-5p mimics group were lower than those in the mimics-NC group(P＜0.05),and they were higher in the THBS3-OE group than those in the control group and the OE-NC group(P＜0.05).The area of cells in the THBS3-OE+miR-363-5p mimics group was larger than that in the OE-NC+miR-363-5p mimics(P＜0.05).The relative expressions of ANP,BNP and β-MHC in the THBS3-OE+miR-363-5p mimics group were higher than those in the OE-NC+miR-363-5p mimics group(P＜0.05).Conclusion Overexpression of miR-363-5p alleviates the pro-hypertrophic effect of AngⅡ on AC16 cells,which is related to the inhibition of THBS3 expression.
维普
万方数据
