长链非编码RNA NONHSAT069381对心肌细胞凋亡的影响及其相关作用机制探究

Effect of long non-coding RNA NONHSAT069381 on cardiomyocyte apoptosis and its related mechanism

目的:探究长链非编码RNA(lncRNA)NONHSAT069381对心肌细胞凋亡的影响及其相关作用机制。方法:通过基因芯片从健康老年和青年志愿者的血液中筛选出与衰老差异性表达的lncRNAs和miRNAs,并通过生物信息学分析确定NONHSAT069381、hsa-miR-124-5p及CASP3为研究对象。使用慢病毒构建NONHSAT069381过表达或敲低的AC16人心肌细胞系,并应用Western blot探究NONHSAT069381对Caspase-3表达的影响,TUNEL染色及Caspase-3、Caspase-8、Caspase-9活性检测评价lncRNA对缺氧复氧诱导的心肌细胞凋亡的影响。双荧光素酶基因报告系统探究hsa-miR-124-5p与NONHSAT069381及CASP3之间的相互作用,并在AC16细胞中验证其作用机制。结果:芯片结果及RT-PCR证实NONHSAT069381及hsa-miR-124-5p在老年组中表达增加(P<0.05)。细胞功能实验结果表明过表达NONHSAT069381促进Caspase-3的表达显著上升(P<0.05)和缺氧复氧诱导的心肌细胞Caspase-3活性增加(P<0.05)及TUNEL阳性细胞数目增加(P<0.05)。双荧光素酶基因报告实验结果证实hsa-miR-124-5p能够分别与NONHSAT069381与CASP3的3’-UTR靶向结合,qRT-PCR和Western Blot结果显示过表达hsa-miR-124-5p后AC16细胞中Caspase-3表达水平下降(P<0.05),且hsa-miR-124-5p的表达并不影响NONHSAT069381的表达水平。结论:lncRNA(NONHSAT069381)通过发挥ceRNA的功能抑制hsa-miR-124-5p而增强Caspase-3表达从而促进影响心肌细胞凋亡。

Objective:To explore the effect of long-chain non-coding RNA(lncRNA)NONHSAT069381 on cardiomyocyte apoptosis and its mechanism.Methods:The differentially expressed lncRNAs and miRNAs,were screened from the blood of healthy elderly and young volunteers by gene chip,and NONHSAT069381,hsa-miR-124-5 p and CASP3 were identified by bioinformatics analysis.Lentivirus was used to construct AC16 human cardiomyocyte line with NONHSAT069381 overexpression or knockdown,and Western blot was used to explore the effect of NONHSAT069381 on Caspase-3 expression.TUNEL staining and Caspase-3,Caspase-8,Caspase-9 activity detection were used to evaluate the effect of lncRNA on cardiomyocyte apoptosis induced by hypoxia-reoxygenation.The interaction between hsa-miR-124-5 p,NONHSAT069381 and CASP3 was explored by double luciferase gene reporting system,and its mechanism was verified in AC16 cells.Results:The result of microarray and RT-PCR showed that the expression of NONHSAT069381 and hsa-miR-124-5 p increased in the elderly group(P<0.05).The result of cell function test showed that overexpression of NONHSAT069381 significantly increased the expression of Caspase-3(P<0.05)and increased the activity of Caspase-3(P<0.05)and the number of TUNEL positive cells in cardiomyocytes(P<0.05)induced by hypoxia-reoxygenation.The result of double luciferase gene reporting assay showed that hsa-miR-124-5 p could bind to the 3’-UTR of NONHSAT069381 and CASP3 respectively.The result of qRT-PCR and Western Blot showed that the expression of Caspase-3 in AC16 cells decreased(P<0.05)after overexpression of hsa-miR-124-5 p,and the expression of hsa-miR-124-5 p did not affect the expression of NONHSAT069381.Conclusions:lncRNA(NONHSAT069381) promotes cardiomyocyte apoptosis by inhibiting hsa-miR-124-5 p and increasing the expression of Caspase-3 by exerting the function of ceRNA.