miR-19a对缺氧复氧诱导心肌细胞SOCS3/JAK1/STAT3信号通路及自噬的影响

The Effects of MiR-19a on the SOCS3/JAK1/STAT3 Signal Pathway and Autophagy Induced by Hypoxia/Reoxygenation in Cardiomyocyte

目的观察miR-19a对缺氧(H)/复氧(R)诱导的大鼠H9c2心肌细胞自噬的影响。方法体外培养大鼠H9c2心肌细胞,建立H/R模型,随机分为Control组、H/R组、H/R-miR-19a阴性对照(NC)组和H/R-miR-19a模拟(mimics)组。实时荧光定量聚合酶链式反应(RT-qPCR)检测转染后各组细胞miR-19a mRNA表达情况;细胞计数试剂盒8(CCK-8)法检测各组细胞增殖情况;膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)染色法检测各组细胞凋亡情况;蛋白免疫印迹法检测各组细胞微管相关蛋白轻链3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ)、核孔蛋白62(p62)、Beclin-1、细胞因子信号转导抑制蛋白3(SOCS3)蛋白表达情况及蛋白质酪氨酸激酶-1(JAK1)及转录活化蛋白3(STAT3)及JAK1、STAT3磷酸化水平。结果与Control组比较,H/R组H9c2心肌细胞miR-19a表达水平、细胞存活率、p62蛋白表达水平、JAK1及STAT3蛋白磷酸化水平降低(P<0.05),细胞凋亡率、Beclin-1、LC3Ⅱ/Ⅰ比值及SOCS3蛋白表达水平升高(P<0.05);与H/R组和H/R-miR-19a NC组比较,H/R-miR-19a mimics组H9c2心肌细胞miR-19a表达水平、细胞存活率、p62蛋白表达水平、p-JAK1及p-STAT3水平升高(P<0.05),凋亡率、Beclin-1、LC3Ⅱ/Ⅰ比值及SOCS3蛋白表达水平降低(P<0.05)。结论过表达miR-19a可抑制H/R诱导的心肌细胞凋亡,可能通过抑制SOCS3表达,激活JAK1/STAT3信号通路抑制H/R诱导的心肌细胞自噬。

Objective To observe the effect of miR-19a on autophagy in rat H9c2 cardiomyocytes induced by hypoxia(H)/reoxygenation(R).Methods The rat H9c2 cardiomyocytes were cultured in vitro to establish the H/R model,which were randomly divided into Control group,H/R group,H/R-miR-19a negative control(NC)group and H/R-miR-19a mimics(mimics)group.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-19a mRNA in each group of cells after transfection;cell counting kit 8(CCK-8)method was used to detect the changes of cell proliferation in each group;Annexin V-Fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI)staining method was used to detect the apoptosis of cells in each group;Western Blot analysis was used to detect the microtubule-associated protein light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ),nucleoporin 62(p62),Beclin-1,inhibitor of cytokine signal transduction protein 3(SOCS3)protein expression,phosphorylation of protein tyrosine kinase-1(JAK1)and transcriptional activator protein 3(STAT3).Results Compared with the control group,the expression of miR-19a,cell survival rate,p62 protein,p-JAK1 and p-STAT3 protein of H9c2 cardiomyocytes in the H/R group were decreased(P<0.05),the apoptosis rate,Beclin-1,LC3Ⅱ/Ⅰratio and SOCS3 protein expression were increased(P<0.05).Compared with H/R group and H/R-miR-19a NC group,the expression of miR-19a,cell survival rate,p62 protein,p-JAK1 and p-STAT3 protein of H9c2 cardiomyocytes in the H/R-miR-19a mimics group were increased(P<0.05),the apoptosis rate,Beclin-1,LC3Ⅱ/Ⅰratio and SOCS3 protein expression were decreased(P<0.05).Conclusion Overexpression of miR-19a could inhibit H/R-induced cardiomyocyte apoptosis,possibly by inhibiting SOCS3 expression and activating JAK1/STAT3 signaling pathway to inhibit H/R-induced cardiomyocyte autophagy.