piRNA-514对心肌细胞肥大的调控作用及其机制

REGULATORY EFFECT OF PIWI-INTERACTING RNA-514 ON CARDIOMYOCYTE HYPERTROPHY AND ITS MECHANISM

目的探讨piwi互作RNA-514(piRNA-514)对心肌细胞肥大的调控作用及其机制。方法采用实时定量PCR(qPCR)检测8周龄雄性C57小鼠肾、肝、脾、肺和心脏中的piRNA-514相对表达量;采用qPCR检测异丙肾上腺素(ISO)分别诱导乳鼠原代心肌细胞0、8、12、24 h后细胞中piRNA-514以及心肌肥厚标记物心房肽(ANF)的相对表达量;采用qPCR检测piRNA-514的类似物agomir-piR-514转染进原代心肌细胞后,细胞中piRNA-514、ANF和脑利钠肽(BNP)的相对表达量,采用鬼笔环肽(phalloidin)和DAPI对原代心肌细胞染色,测定细胞表面积;采用qPCR检测agomir-piR-514转染进原代心肌细胞,经ISO诱导24 h后细胞中piRNA-514、ANF和β-肌球蛋白重链(β-MHC)的相对表达量,phalloidin和DAPI对原代心肌细胞染色,激光共聚焦显微镜观察细胞表型变化并测定细胞表面积。结果qPCR方法检测结果显示,与肾、肝、脾、肺相比,piRNA-514在心脏中的表达量最高(F=24.47,P<0.01);qPCR方法检测结果显示,随着ISO诱导时间延长,原代心肌细胞中piRNA-514表达水平逐渐降低(F=12.26,P<0.01),ANF表达水平逐渐升高(F=4.49,P<0.01);qPCR方法检测结果显示,与对照组相比,转染agomir-piR-514组的心肌细胞中piRNA-514的相对表达量显著增高,ANF以及BNP的相对表达量显著降低(F=7.91~14.25,t=14.84~29.43,P<0.05),对细胞的表面积测定结果显示,转染ago-mir-piR-514组的细胞表面积与对照组相比明显减小(F=36.12,t=8.73,P<0.05);qPCR方法检测结果显示,与ISO组相比,agomir-piR-514+ISO组的原代心肌细胞中piRNA-514相对表达量显著增高,ANF和β-MHC的相对表达量显著降低(F=10.89~21.57,t=6.94~12.96,P<0.05),细胞表面积测定结果显示,agomir-piR-514+ISO组的细胞表面积与ISO组相比显著减小(F=23.82,t=16.41,P<0.05)。结论piRNA-514具有调控心肌细胞肥大的功能,过表达piRNA-514可以抑制ISO诱导的心肌细胞肥大。

Objective To investigate the regulatory effect of piwi-interacting RNA-514(piRNA-514)on cardiomyocyte hypertrophy and its mechanism.Methods Real-time quantitative PCR(qPCR)was used to measure the relative expression of piRNA-514 in the kidney,liver,spleen,lung,and heart of male C57 mice aged 8 weeks;qPCR was used to measure the relative expression of piRNA-514 and the marker for myocardial hypertrophy atrial natriuretic factor(ANF)in primary cardiomyocytes of neonatal mice induced by isoproterenol(ISO)for 0,8,12,and 24 h;qPCR was used to measure the relative expression of piRNA-514,ANF,and brain natriuretic peptide(BNP)in primary cardiomyocytes transfected with the piRNA-514 analogue agomir-piR-514,the primary cardiomyocytes were stained with phalloidin and 4′,6-diamidino-2-phenylindole(DAPI),and cell surface area was measured;qPCR was used to measure the relative expression of piRNA-514,ANF,andβ-myosin heavy chain(β-MHC)in primary cardiomyocytes transfected with agomir-piR-514 and induced by ISO for 24 h,the primary cardiomyocytes were stained with phalloidin and DAPI,phenotypic changes of cardiomyocytes were observed under a laser confocal microscope,and cell surface area was measured.Results The results of qPCR showed that compared with the kidney,liver,spleen,and lung,the heart showed that highest peak of the expression of piRNA-514(F=24.47,P<0.01).Over the time of ISO induction,there was a gra-dual reduction in the expression of piRNA-514(F=12.26,P<0.01)and a gradual increase in the expression of ANF(F=4.49,P<0.01)in primary cardiomyocytes.The results of qPCR showed that compared with the control group,the agomir-piR-514 group had a significant increase in the relative expression of piRNA-514 and significant reductions in the relative expression of ANF and BNP in cardiomyocytes(F=7.91-14.25,t=14.84-29.43,P<0.05),and the measurement of cell surface area showed that the agomir-piR-514 group had a significantly smaller cell surface area than the control group(F=36.12,t=8.73,P<0.05).The results of qPCR showed that compared with the ISO group,the ago-mir-piR-514+ISO group had a significant increase in the relative expression of piRNA-514 and significant reductions in the relative expression of ANF andβ-MHC in primary cardiomyocytes(F=10.89-21.57,t=6.94-12.96,P<0.05),and the measurement of cell surface area showed that the agomir-piR-514+ISO group had a significantly smaller cell surface area than the ISO group(F=23.82,t=16.41,P<0.05).Conclusion piRNA-514 has the function of regulating cardiomyocyte hypertrophy,and overexpression of piRNA-514 can inhibit cardiomyocyte hypertrophy induced by ISO.