lncRNA MALAT1通过调控miR-181a促进氧化应激模型中的心肌细胞损伤

LncRNA MALAT1 promoted myocardial cell damage in oxidative stress models by regulating miR-181a

目的在心肌细胞氧化应激模型中探究长链非编码RNA(lncRNA)转移相关肺腺癌转录物1(MALAT1)与微小RNA-181a(miR-181a)的表达及作用机制。方法qRT-PCR检测30例急性心肌梗死患者(AMI组)和30例健康对照组(Normal组)外周血中MALAT1与miR-181a的表达,Pearson相关性分析明确MALAT1与miR-181a在AMI中的相关性;lncBase在线预测数据库预测MALAT1与miR-181a之间的结合位点,并利用双荧光素酶基因报告实验进行验证;采用过氧化氢(H_(2)O_(2))处理人心肌细胞株AC16建立心肌细胞氧化应激模型,将沉默MALAT表达的siRNA(si-MALAT)和阴性对照si-NC转染入AC16细胞中,并将细胞分为:H_(2)O_(2)处理(H_(2)O_(2))组,H_(2)O_(2)+si-NC组,H_(2)O_(2)+si-MALAT组;CCK-8法检测各组细胞的增殖活性,TUNEL法检测细胞凋亡情况,Western blot实验检测各组细胞中促凋亡蛋白裂解型半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)、Bcl-2相关X蛋白(Bax)和凋亡抑制蛋白B细胞淋巴瘤/白血病-2(Bcl-2)的表达水平。结果与Normal组相比,AMI组患者MALAT1的表达水平升高,而miR-181a的表达水平降低(P<0.05),且MALAT1和miR-181a表达呈负相关。lncBase在线预测数据库预测和双荧光素酶基因报告实验表明MALAT1可靶向调控miR-181a表达。与H_(2)O_(2)组相比,H_(2)O_(2)+si-MALAT1组细胞活力升高(P<0.05),TUNEL阳性率降低(P<0.05),cleaved caspase-3和Bax表达水平降低(P<0.05),而Bcl-2的表达水平升高(P<0.05),H_(2)O_(2)+si-NC组均无明显改变(P>0.05)。结论LncRNA MALAT1在AMI患者中表达升高,可通过靶向抑制miR-181a促进氧化应激诱导的心肌细胞损伤。

Objective To investigate the expression and mechanism of long non-coding RNA(lncRNA)metastasis-associated lung adenocarcinoma transcript 1(MALAT1)and microRNA-181a(miR-181a)in a myocardial cell oxidative stress model.Methods The expression of MALAT1 and miR-181a in peripheral blood of 30 patients with acute myocardial infarction(AMI group)and 30 healthy controls(Normal group)was detected by qRT-PCR.Pearson correlation analysis was performed to determine the correlation between MALAT1 and miR-181a in AMI.The binding sites between MALAT1 and miR-181a were predicted using the lncBase online prediction database and validated by dual-luciferase reporter assay.An oxidative stress model of myocardial cells was established by hydrogen peroxide(H_(2)O_(2))treatment in AC16 human myocardial cell line.siRNA targeting MALAT1(si-MALAT)and negative control siRNA(si-NC)were transfected into AC16 cells,and the cells were divided into H_(2)O_(2) treatment(H_(2)O_(2))group,H_(2)O_(2)+si-NC group,and H_(2)O_(2)+si-MALAT group.Cell proliferation activity was detected by CCK-8 assay,cell apoptosis was assessed by TUNEL assay,and the expression levels of cleaved caspase-3,Bcl-2-associated X protein(Bax),and B-cell lymphoma-2(Bcl-2)were determined by Western blot.Results Compared to the Normal group,the expression of MALAT1 was upregulated and the expression of miR-181a was downregulated in the AMI group(P<0.05),and there was a negative correlation between MALAT1 and miR-181a expression.The lncBase online prediction database and dual-luciferase reporter assay results had proven that MALAT1 could target and regulate the expression of miR-181a.Compared to the H_(2)O_(2) group,the H_(2)O_(2)+si-MALAT group showed increased cell viability(P<0.05),decreased TUNEL-positive rate(P<0.05),decreased expression levels of cleaved caspase-3 and Bax(P<0.05),and increased expression level of Bcl-2(P<0.05),while the H_(2)O_(2)+si-NC group showed no significant changes(P>0.05).Conclusion LncRNA MALAT1 expression is elevated in AMI patients,which could promote oxidative stress-induced myocardial cell damage through targeted inhibition of miR-181a.