摘要
目的研究microRNA-9(miR-9)表达对肿瘤坏死因子α(TNF-α)诱导心肌细胞(H9c2)损伤的影响,并探讨其分子机制。方法使用CCK8试剂盒检测不同浓度(0、5、10、15和20μg/mL)TNF-α刺激H9c2或TNF-α作用于H9c2不同时间(0、12、24、48和72 h)后心肌细胞活性;向H9c2细胞中转染miR-9抑制剂(miR-9-inhibitor)以敲低miR-9的表达,通过实时荧光定量PCR法检测miR-9的表达,通过免疫印迹法检测SIRT1、p65及p-p65蛋白表达;荧光素酶基因报告系统用于检测miR-9与SIRT1靶向结合情况。结果 TNF-α以剂量和时间依赖性降低心肌细胞活性和促进miR-9在心肌细胞中的表达。与miR-9未敲低组相比,TNF-α刺激后,miR-9敲低组H9c2细胞活性更高(P<0.05)。荧光素酶基因报告结果显示:miR-9靶向抑制SIRT1的表达,miR-9敲低可显著促进H9c2细胞中SIRT1蛋白的表达,显著降低p-p65/p65的比例(P均<0.05)。结论 miR-9通过抑制SIRT1促进NF-κB通路激活。敲低miR-9可抑制NF-κB通路激活从而减轻TNF-α诱导的心肌细胞损伤,提高心肌细胞活性。
Objective To study the influence of expression of microRNA-9(miR-9)on injury of cardiomyocytes(H9 c2)induced by tumor necrosis factor-α(TNF-α),and discuss possible molecular mechanism.Methods H9 c2 activity was detected by using CCK8 kit after TNF-αstimulated H9 c2 in different doses(0μg/ml,5μg/ml,10μg/ml,15μg/ml and 20μg/ml)or at different time points(0 h,12 h,24 h,48 h and 72 h).The expression of miR-9 was knocked down through transfected miR-9-inhibitor to H9 c2.The expression of miR-9 was detected by using qRT-PCR,and protein expressions of SIRT1,p65 and p-p65 were detected by using Western blotting assay.The targeted binding of miR-9 and SIRT1 was detected by using luciferase reporter gene assay.Results The expression of miR-9 in miR-9 was reduced by TNF-αin a dose-dependent manner and a time-dependent manner.Compared with miR-9 non-knockdown group,H9 c2 activity was higher in miR-9 knockdown group after TNF-αstimulation(P<0.05).The results of luciferase reporter gene assay showed that miR-9 targeted to inhibit SIRT1 expression,and miR-9 knockdown significantly improved SIRT1 protein expression and decreased ratio of p-p65 to p65 in H9 c2(all P<0.05).Conclusion MiR-9 can improve NF-κB pathway activation through inhibiting SIRT1.MiR-9 knockdown can inhibit NF-κB pathway activation to relieve cardiomyocyte injury induced by TNF-αand promote cardiomyocyte activity.
作者
张朝华
刘旭帮
朱银川
汤建民
Zhang Chaohua;Liu Xubang;Zhu Yinchuan;Tang Jianmin(Department of Cardiovascular Medicine,Zhengzhou People's Hospital,Zhengzhou 450000,China)
出处
《中国循证心血管医学杂志》
2021年第7期803-806,共4页
Chinese Journal of Evidence-Based Cardiovascular Medicine
基金
河南省2018年度科技攻关计划项目(182102310166)。
作者简介
通讯作者:汤建民,E-mail:zhangchaohua2508@126.com。
