miR-27a-3p通过靶向Sema7A调控病毒性心肌炎心肌细胞损伤的机制研究

目的探讨微小RNA-27a-3p(miR-27a-3p)过表达对病毒性心肌炎(VMC)细胞损伤的影响及其可能的作用机制。方法原代培养大鼠心肌细胞,柯萨奇B3病毒(CVB3)感染心肌细胞建立VMC模型(CVB3组)。正常心肌细胞作为对照组,分别将miR-NC、miR-27a-3p mimics、si-NC,si-Sema7A分别转染至CVB3感染的心肌细胞,记为CVB3+miR-NC组、CVB3+miR-27a-3p组、CVB3+si-NC组、CVB3+si-Sema7A组。分别将miR-27a-3p mimics与pcDNA,miR-27a-3p mimics与pcDNA-Sema7A共转染至CVB3感染的心肌细胞,记为CVB3+miR-27a-3p+pcDNA组、CVB3+miR-27a-3p+pcDNA-Sema7A组。采用实时荧光定量聚合酶链反应(qRT-PCR)与Western blot法分别检测细胞中miR-27a-3p、信号蛋白7A(Sema7A)的表达水平;流式细胞术检测miR-27a-3p过表达或干扰Sema7A表达后心肌细胞的凋亡率;酶联免疫吸附(ELISA)法检测细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)的含量;双荧光素酶报告基因验证miR-27a-3p与Sema7A的靶向关系;Western blot检测B淋巴细胞瘤-2相关蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)、B淋巴细胞瘤-2(Bcl-2)蛋白表达。两组间比较采用独立样本t检验。结果与对照组比较,CVB3组miR-27a-3p的表达水平(1.01±0.09比0.42±0.04)降低,Sema7A的mRNA和蛋白表达水平(1.02±0.09比2.15±0.21、0.43±0.04比0.94±0.09)升高,TNF-α、IL-1水平(403.56±38.16)pg/mL比(1156.48±63.59)pg/mL>(29.45±3.54)pg/mL比(136.57±12.47)pg/mL]升高,细胞凋亡率(5.36±0.54)%比(24.58±2.14)%]升高,Bax、cleaved caspase-3的表达水平升高,Bcl-2的表达水平降低(P均<0.05);与CVB3+miR-NC组比较,CVB3+miR-27a-3p组TNF-α,IL-1水平(1187.69±71.42)pg/mL比(516.28±48.31)pg/mL、(147.25±14.05)pg/mL比(43.68±5.02)pg/mL]和细胞凋亡率(26.38±2.55)%比(10.24±1.15)%]降低,Bax、cleaved caspase-3表达水平降低,Bcl-2表达水平升高(P均<0.05);双荧光素酶报告实验证实miR-27a-3p可靶向调控Sema7A的表达;Sema7A过表达可逆转miR-27a-3p过表达对CVB3诱导的VMC细胞损伤的作用。结论miR-27a-3p过表达可降低VMC细胞中炎症因子的表达及抑制细胞凋亡从而减轻心肌损伤,其作用机制可能与靶向调控Sema7A的表达有关。

Overexpression of miR-27a-3p inhibits myocardial cell injury in viral myocarditis by targeting Sema7A

Objective To investigate the effect of overexpressed microRNA-27a-3p (miR-27a-3p) on viral myocarditis (VMC) cell injury and its mechanisms.Methods Primary cardiomyocytes of rats were infected with Coxsackie B3 virus (CVB3) to establish VMC cell model (CVB3 group).Normal cardiomyocytes as control (con),and the miR-NC,miR-27a-3p mimics,si-NC,and si-Sema7A were transfected into CVB3-infected cardiomyocytes respectively,which were named as CVB3+miR-NC,CVB3+miR-27a-3p,CVB3+si-NC,and CVB3+si-Sema7A group.The miR-27a-3p mimics and pcDNA,miR-27a-3p mimics and pcDNA-Sema7A were co-transfected into CVB3-infected cardiomyocytes,respectively,and named as CVB3+miR-27a-3p+pcDNA and CVB3+miR-27a-3p+pcDNA-Sema7A group.Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression levels of miR-27a-3p and Sema7A in cells.Flow cytometry was used to detect the apoptotic rate of cardiomyocytes after overexpression of miR-27a-3p or knock-down Sema7A.The levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1 in the cells were detected by enzyme-linked immunosorbent assay (ELISA).The targeting relationship between miR-27a-3p and Sema7A was verified by dual luciferase reporter gene assay.The expression of Bax,cleaved caspase-3 and Bcl-2 protein was detected by Western blot.Results Compared with Con group,the expression level of miR-27a-3p in CVB3 group was significantly decreased (1.01±0.09 vs 0.42±0.04,P < 0.05),and the mRNA and protein levels of Sema7A were significantly increased (1.02±0.09 vs 2.15±0.21,P < 0.05,0.43±0.04 vs 0.94±0.09,P < 0.05),the levels of TNF-α and IL-1 were significantly increased(403.56±38.16)pg/mL vs (1156.48±63.59)pg/mL,(29.45±3.54)pg/mL vs(136.57±12.47) pg/ mL,P < 0.05],the apoptosis rate was increased significantly(5.36±0.54 vs 24.58±2.14,P < 0.05),the expression levels of Bax and cleaved caspase-3 were increased significantly (P < 0.05),and the expression level of Bcl-2 was decreased significantly (P < 0.05).Compared with the CVB3+miR-NC group,the levels of TNF-α and IL-1 in the CVB3+miR-27a-3p group were significantly reduced(1187.69±71.42)pg/mL vs (516.28±48.31) pg/mL,(147.25±14.05) pg/ mL vs (43.68±5.02) pg/ mL,P < 0.05],the apoptosis rate was decreased(26.38±2.55)% vs (10.24±1.15)%,P < 0.05],the expression levels of Bax and cleaved caspase-3 were decreased (P < 0.05),and the expression level of Bcl-2 was increased (P < 0.05).Double luciferase reporting experiments confirmed that miR-27a-3p could target Sema7A expression.Sema7A overexpression could reverse the effect of miR-27a-3p overexpression on CVB3-induced VMC cell injury.Conclusion Overexpression of miR-27a-3p can reduce the expression of inflammatory factors and inhibit apoptosis in VMC cells,thereby reducing myocardial injury.The mechanism may be related to the targeted regulation of the expression of Sema7A.