piRNA-5938可调控心肌细胞凋亡和线粒体分裂

piRNA-5938 can regulate cardiomyocyte apoptosis and mitochondrial fission

背景:心肌细胞凋亡在心肌缺血再灌注损伤中扮演了重要角色。最新研究表明生殖系统调控分子PIWI相互作用RNA(PIWI-Interacting RNA,piRNA)参与了心脏病理过程调控,然而在心肌细胞凋亡中的作用尚未阐明,尤其涉及线粒体途径的机制尚不清楚。目的:探究piRNA-5938对心肌细胞凋亡和线粒体分裂的调节作用及分子机制。方法:深度测序检测正常和缺血再灌注模型小鼠及大鼠心脏中piRNA的表达丰度,实时荧光定量PCR法验证结果。分别用双氧水和缺氧再复氧条件构建心肌细胞凋亡模型,实时荧光定量PCR法检测piRNA-5938的表达水平。合成piPNA-5938特异性拮抗剂转染心肌细胞,再用双氧水处理,TUNEL法检测细胞凋亡情况,MitoTracker法检测线粒体分裂情况。RNAhybrid软件预测与piRNA-5938相互作用的microRNA。结果与结论:①与假手术组比,缺血再灌注小鼠心脏中422种piRNA表达水平改变,其中289条上调,133条下调;piRNA-5938的表达显著增加(P<0.01);②双氧水诱导心肌细胞凋亡后piRNA-5938的表达水平显著上调,且呈浓度依赖性(P<0.05);③缺氧再复氧诱导心肌细胞凋亡后piRNA-5938表达显著增加(P<0.01);④与阴性对照组比,敲低piRNA-5938后,双氧水诱导的心肌细胞凋亡率明显降低(P<0.05);⑤敲低piRNA-5938后,双氧水诱导的心肌细胞线粒体分裂明显得到改善(P<0.05);⑥piRNA-5938与线粒体分裂调控分子miR-324和miR-668序列能很好地互补配对,且具有高度保守性;⑦提示piRNA-5938调控了心肌细胞凋亡和线粒体分裂,并且可能通过miR-324或miR-668发挥作用。

BACKGROUND:Cardiomyocyte apoptosis plays a critical role in ischemia/reperfusion injury.Recent studies have shown that PIWI-Interacting RNA(piRNA),a reproductive system regulator,participates in the pathogenesis of cardiac disorder.However,the role of piRNA in cardiomyocyte apoptosis has not been clarified;especially the mitochondria-dependent pathway involved is poorly understood.OBJECTIVE:To study the role and molecular mechanism of piRNA-5938 in cardiomyocyte apoptosis and mitochondrial fission.METHODS:The abundance of piRNA in normal and ischemic/reperfusion hearts of mice and rats was detected by deep sequencing,and the results were verified by real-time fluorescence quantitative PCR.Cardiac apoptosis was induced by hydrogen peroxide or anoxia/reoxygenation,in which the expression level of piRNA-5938 was detected by real-time fluorescence quantitative PCR.piRNA-5938 antagomir was synthesized and transfected into cardiomyocytes and then the cells were treated with hydrogen peroxide.Apoptosis was detected by TUNEL and mitochondrial division was evaluated by MitoTracker.RNAhybrid software was used to predict microRNAs that interact with piRNA-5938.RESULTS AND CONCLUSION:Compared with the sham group,the expression levels of 422 piRNAs were changed in the mouse ischemic/reperfusion heart,of which 289 were up-regulated and 133 were down-regulated,and piRNA-5938 expression increased significantly(P<0.01).The expression level of piRNA-5938 was significantly up-regulated after cardiomyocyte apoptosis induced by H2O2 in a concentration-dependent manner(P<0.05).piRNA-5938 was significantly increased after anoxia/reoxygenation-induced cardiomyocyte apoptosis(P<0.01).Compared with the negative control group,knockdown of piRNA-5938 significantly reduced the apoptosis rate of cardiomyocytes induced by hydrogen peroxide(P<0.05).The mitochondrial fission induced by hydrogen peroxide was significantly inhibited by the knockdown of piRNA-5938(P<0.05).piRNA-5938 was highly conserved in complementation with mitochondrial fission regulators mir-324 and Mir-668.To conclude,piRNA-5938 regulates cardiomyocyte apoptosis and mitochondrial fission,which probably functions through targeting mir-324 or mir-668.