miR-217-5p在病毒性心肌炎患者血清中的表达变化及其对CVB3诱导的心肌细胞损伤影响

The expression change of miR-217-5p in the serum of patients with viral myocarditis and its effect on CVB3-induced myocardial cell injury

目的探讨微小RNA-217-5p(miR-217-5p)在病毒性心肌炎(VMC)患者血清中的表达变化及其对柯萨奇病毒B3(CVB3)诱导的心肌细胞损伤的影响。方法回顾性选取77例VMC患者为研究对象,同期77例健康体检者为对照。将大鼠H9c2心肌细胞分为Con组(未做任何处理)、CVB3组(含CVB3的DMEM处理)、CVB3+miR-217-5p组(H9c2细胞转染miR-217-5p模拟物后经含CVB3的DMEM处理)、CVB3+pcDNA-SIRT1组[H9c2细胞转染pcDNA-沉默信息调节因子2同源蛋白1(SIRT1后经含CVB3的DMEM处理)]、CVB3+miR-217-5p+si-SIRT1组(H9c2细胞转染miR-217-5p+si-SIRT1后经含CVB3的DMEM处理)。采用实时定量PCR(qRT-PCR)检测miR-195-5p和SIRT1 mRNA在VMC患者及健康体检者血清中的表达水平;双荧光素酶报告实验验证miR-217-5p、SIRT1靶向关系;流式细胞术测定细胞凋亡率、Western blot检测细胞SIRT1、活化型半胱氨酸蛋白酶3(cleaved-caspase3)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)表达水平;酶联免疫吸附法(ELISA)检测细胞分泌肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、IL-8水平。结果miR-217-5p在病VMC患者血清中表达水平较健康体检者高,SIRT1 mRNA表达水平较健康体检者低(P<0.05)。miR-217-5p可靶向调控SIRT1蛋白的表达。与Con组相比,CVB3组心肌细胞中miR-217-5p表达水平、细胞凋亡率、cleaved-caspase3、Bax蛋白表达水平、TNF-α、IL-1β、IL-6、IL-8水平升高,Bcl-2蛋白表达水平降低(P<0.05)。与CVB3组相比,CVB3+anti-miR-217-5p组miR-217-5p表达水平、细胞凋亡率、cleaved-caspase3、Bax蛋白表达水平、TNF-α、IL-1β、IL-6、IL-8水平降低,SIRT1、Bcl-2蛋白表达水平升高(P<0.05);与CVB3组相比,CVB3+pcDNA-SIRT1组细胞凋亡率、cleaved-caspase3、Bax蛋白表达水平、TNF-α、IL-1β、IL-6、IL-8水平降低,SIRT1、Bcl-2蛋白表达水平升高(P<0.05);与CVB3+miR-217-5p组相比,CVB3+anti-miR-217-5p+si-SIRT1组细胞凋亡率、cleaved-caspase3、Bax蛋白表达水平、TNF-α、IL-1β、IL-6、IL-8水平升高,SIRT1、Bcl-2蛋白表达水平降低(P<0.05)。结论miR-217-5p可能通过靶向调控SIRT1蛋白的表达进一步调控CVB3诱导的心肌细胞损伤。

Objective To investigate the expression changes of microRNA-217-5p(miR-217-5p)in the serum of patients with viral myocarditis(VMC)and its effect on the myocardial cell injury induced by coxsackievirus B3(CVB3).Methods A retrospective study was conducted on 77 patients with VMC and 77 healthy controls.Rat H9c2 cardiomyocytes were divided into Con group(no treatment),CVB3 group(treated with DMEM containing CVB3),CVB3+miR-217-5p group(treated with DMEM containing CVB3 after H9c2 cells were transfected with miR-217-5p mimics),CVB3+pcDNA-SIRT1 group(treated with DMEM containing CVB3 after H9c2 cells were transfected with pcDNA-silencing information regulator 2 homolog 1(SIRT1)),and CVB3+miR-217-5p+si-SIRT1 group(treated with DMEM containing CVB3 after H9c2 cells were transfected with miR-217-5p+si-SIRT1).Real-time quantitative PCR(qRT-PCR)was used to detect the expression levels of miR-195-5p and SIRT1 mRNA in the serum of VMC patients and healthy controls.Double luciferase reporter assay was used to verify the targeted relationship between miR-217-5p and SIRT1.Flow cytometry was used to measure the apoptosis rate of cells,and Western blot was used to detect the expression levels of SIRT1,activated caspase3,Bcl-2,and Bax.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6,and IL-8 levels in cells.Results The expression level of miR-217-5p in the serum of patients with VMC was higher than that of healthy subjects,and the expression level of SIRT1 mRNA was lower than that of healthy subjects(P<0.05).miR-217-5p can target and regulate the expression of SIRT1 protein.Compared with the CVB3 group,the expression level of miR-217-5p,the apoptosis rate,the expression levels of cleaved-caspase3 and Bax protein,the levels of TNF-α,IL-1β,IL-6,and IL-8 increased,and the expression level of Bcl-2 protein decreased in the CVB3+anti-miR-217-5p group(P<0.05).Compared with the CVB3 group,the expression level of miR-217-5p,the apoptosis rate,the expression levels of cleaved-caspase3 and Bax protein,the levels of TNF-α,IL-1β,IL-6,and IL-8 decreased,and the expression levels of SIRT1 and Bcl-2 protein increased in the CVB3+pcDNA-SIRT1 group(P<0.05).Compared with the CVB3+miR-217-5p group,the apoptosis rate,the expression levels of cleaved-caspase3 and Bax protein,the levels of TNF-α,IL-1β,IL-6,and IL-8 increased,and the expression levels of SIRT1 and Bcl-2 protein decreased in the CVB3+anti-miR-217-5p+si-SIRT1 group(P<0.05).Conclusion miR-217-5p may further regulate the myocardial cell injury induced by CVB3 by targeting and regulating the expression of SIRT1 protein.