miR-652-3p靶向同源异型核基因1对血管紧张素Ⅱ诱导的心肌细胞凋亡的影响

Effects of miR-652-3p targeting homeotype nuclear gene 1 in the regulation of angiotensin Ⅱ-induced cardiomyocyte apoptosis

目的探讨miR-652-3p靶向同源异型核基因1(PRRX1)对血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞凋亡的影响。方法大鼠心肌细胞H9c2细胞采用正常培养基培养为对照组细胞,用含1μmol/L AngⅡ的培养基培养为AngⅡ组细胞;分别转染miR-652-3p阳性对照序列(NC)和转染miR-652-3p mimics后用含1μmol/L AngⅡ的培养基培养为AngⅡ+NC组和AngⅡ+miR-652-3p组细胞;将miR-652-3p mimics分别与PRRX1阳性对照质粒和PRRX1过表达质粒转染至H9c2细胞中用含1μmol/L AngⅡ的培养基培养,分别为AngⅡ+miR-652-3p+Vctor组和AngⅡ+miR-652-3p+PRRX1组细胞。实时荧光定量PCR(RT-qPCR)检测H9c2细胞中miR-652-3p表达水平,流式细胞术检测细胞凋亡,用Western blot检测细胞中PRRX1、Bax和Bcl-2蛋白表达水平。双荧光素酶报告基因实验验证H9c2细胞中miR-652-3p与PRRX1调控关系。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果与对照组比较,AngⅡ组H9c2细胞中miR-652-3p水平(1.00±0.08比0.21±0.05)、Bcl-2蛋白水平(0.83±0.08比0.40±0.04)均较低,而PRRX1蛋白水平(0.06±0.01比0.41±0.04)、凋亡率(5.02﹪±1.41﹪比25.33﹪±3.75﹪)、Bax蛋白水平(0.46±0.05比0.96±0.10)均较高,差异具有统计学意义(P均<0.05)。与AngⅡ+NC组比较,AngⅡ+miR-652-3p组H9c2细胞中miR-652-3p的表达水平(0.24±0.06比0.98±0.07)、Bcl-2蛋白水平(0.38±0.04比0.72±0.07)均较高,而PRRX1蛋白水平(0.39±0.04比0.13±0.01)、凋亡率(27.02﹪±4.11﹪比12.19﹪±1.63﹪)、Bax蛋白水平(0.95±0.09比0.53±0.05)均较低,差异具有统计学意义(P均<0.05)。与AngⅡ+miR-652-3p+Vctor组比较,AngⅡ+miR-652-3p+PRRX1组H9c2细胞凋亡率(12.88﹪±1.84﹪比25.45﹪±3.58﹪)、PRRX1蛋白水平(0.13±0.01比0.35±0.04)和Bax蛋白水平(0.54±0.05比0.82±0.08)均较高,差异具有统计学意义(P均<0.05),而Bcl-2蛋白表达水平(0.72±0.07比0.46±0.05)降低,差异具有统计学意义(P<0.05)。结论AngⅡ能够下调心肌细胞中miR-652-3p的表达,上调miR-652-3p可通过靶向抑制PRRX1的表达减少AngⅡ诱导的H9c2细胞凋亡。

Objective To investigate the effect of miR-652-3p targeting autonomous heterologous nuclear gene 1(PRRX1)in the regulation of angiotensinⅡ(AngII)-induced cardiomyocyte apoptosis.Methods Rat cardiac cell H9c2 cultured under normal conditions were used as the control group,while AngⅡgroup were established in medium containing 1μmol/L AngⅡ.These cells were transfected with miR-652-3p+Vctor and miR-652-3p mimics respectively.In addition AngⅡ-H9c2 cells transfected with miR-652-3p mimics were transfected with pc-PRRX1 and overexpressed-PRRX1.The expression of miR-652-3p in H9c2 was determined by quantitative real-time PCR(qRT-qPCR).Detection of cell apoptosis was evaluated by using flow cytometry.In addition the protein expression levels of Bax and Bcl-2 were assessed by Western blot analysis.Gene regulatory relations between miR-652-3p and PRRX1 were confirmed by Dual-Luciferase reporter gene system.The t-test was applied for comparison between two groups and single factor variance analysis(one-way ANOVA)was used for comparison among several groups.The SNK-q test method was used to compare between groups.Results Compared with control group,the level of miR-652-3p(1.00±0.08 vs 0.21±0.05)and Bcl-2 protein(0.83±0.08 vs 0.40±0.04)in H9c2 cells were lower in AngⅡgroup,while PRRX1 protein level(0.06±0.01 vs 0.41±0.04),apoptosis rate(5.02﹪±1.41﹪vs 25.33﹪±3.75﹪),Bax protein level(0.46±0.05 vs 0.96±0.10)were higher(all P<0.05).Compared with AngⅡ+NC group,the expression level of miR-652-3p(0.24±0.06 vs 0.98±0.07)and Bcl-2 protein level in the AngⅡ+miR-652-3p group(0.38±0.04 vs 0.72±0.07)were higher while the PRRX1 protein level(0.39±0.04 vs 0.13±0.01),and apoptosis rate(27.02﹪±4.11﹪vs 12.19﹪±1.63﹪),Bax protein level(0.95±0.09 vs 0.53±0.05)were lower(all P<0.05).Compared with the AngⅡ+miR-652-3p+Vctor group,the apoptosis rate of H9c2 cells(12.88﹪±1.84﹪vs 25.45﹪±3.58﹪)as well as expression of PRRX1 protein(0.13±0.01 vs 0.35±0.04)and Bax protein(0.54±0.05 vs 0.82±0.08)in the AngⅡ+miR-652-3p+PRRX1 group were higher(all P<0.05),whereas Bcl-2 protein level(0.72±0.07 vs 0.46±0.05)was lower(all P<0.05).Conclusion AngⅡcould down-regulatethe expression of miR-652-3p in cardiomyocytes.Additionally,Up-regulation of miR-652-3p could reduce the apoptosis of H9c2 cells induced by AngⅡby targeting the inhibition of PRRX1 expression.