MicroRNA-363-5p靶向血小板反应蛋白-3调控心肌细胞肥大的作用机制研究

MicroRNA-363-5p targets thrombospondin-3 to regulate myocardial hypertrophy

摘要:

目的 探讨microRNA-363-5p（miR-363-5p）靶向血小板反应蛋白-3（THBS3）对心肌肥大的调节作用。方法 体外人心肌细胞（AC16）经血管紧张素Ⅱ（AngⅡ）处理复制心肌肥大体外模型，随后鬼笔环肽染色观察细胞骨架，Western blotting检测心肌肥大体外模型中胚胎期基因的蛋白表达，以确认模型复制的有效性。实时荧光定量聚合酶链反应检测心肌肥大体外模型中miR-363-5p表达。Western blotting检测肥大心肌细胞中转染miR-363-5p mimics和miR-363-5p inhibitor后，肥大相关表型的变化。双荧光素酶报告基因实验验证miR-363-5p与THBS3的3'-UTR结合作用。设计挽救实验，同时过表达THBS3与miR-363-5p，以评估THBS3是否介导miR-363-5p对心肌肥大的调控。结果 AngⅡ组细胞面积较对照组大（P <0.05），心房钠尿肽（ANP）、B型钠尿肽（BNP）、肌球蛋白β重链（β-MHC）及miR-363-5p较对照组高（P <0.05）。miR-363-5p mimics组miR-363-5p相对表达量较mimics-NC组高（P <0.05），miR-363-5p inhibitor组相对表达量较inhibitor-NC组低（P <0.05）；miR-363-5p mimics组ANP、BNP、β-MHC相对表达量较mimics-NC组低（P <0.05），miR-363-5p inhibitor组相对表达量较inhibitor-NC组高（P <0.05）。miR-363-5p mimics组细胞面积较mimics-NC组小（P <0.05），miR-363-5p inhibitor组较inhibitor-NC组大（P <0.05）。miR-363-5p mimics + THBS3-WT组THBS3-WT荧光素酶活性较mimics-NC + THBS3-WT组低。mimics-NC+THBS3-MUT组与miR-363-5p mimics + THBS3-MUT组THBS3-MUT荧光素酶活性比较，差异无统计学意义（P >0.05）。miR-363-5p mimics组THBS3 mRNA和蛋白相对表达量较mimics-NC组低（P <0.05）。THBS3-OE组THBS3 mRNA和蛋白相对表达量较对照组、OE-NC组高（P <0.05）。THBS3-OE + miR-363-5p mimics组细胞面积较OE-NC + miR-363-5p mimics组大（P <0.05）。THBS3-OE + miR-363-5p mimics组ANP、BNP及β-MHC相对表达量较OE-NC + miR-363-5p mimics组高（P <0.05）。结论 过表达miR-363-5p可抑制AngⅡ对AC16细胞的促肥大作用，其机制与减少THBS3表达有关。

Abstract:

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 (AC16) 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 AngII group 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.