microRNA在柯萨奇B3病毒诱导的心肌微血管内皮细胞凋亡中的差异表达  

Differential expression of microRNA in coxsackievirus B_3 induced cardiac microvascular endothelial cell apoptosis

作　　者：虞勇[1] 虞莹[1] 王兴冈[1] 邹云增[1] 陈瑞珍[1] 

机构地区：[1]复旦大学附属中山医院心内科 上海市心血管病研究所 卫生部病毒性心脏病重点实验室,上海200032

出　　处：《中华临床医师杂志（电子版）》2014年第21期94-98,共5页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学基金(31070786)

摘　　要：目的本研究通过体外培养心脏微血管内皮细胞(CMVECs),经柯萨奇B3病毒(CVB3)感染后,利用miRNA寡核苷酸基因芯片技术筛选差异表达的miRNAs,进一步探讨miRNA在CVB3诱导CMVECs凋亡中的潜在作用机制。方法原代分离培养大鼠CMVECs细胞,以100TCID50CVB3病毒感染48 h后检测Caspase-3活性和细胞凋亡;提取CMVECs细胞RNA,用Agilent大鼠miRNA寡核苷酸基因芯片进行检测,并通过TargetScan、miranda、mirbase和mirdb数据库选取出差异表达明显并与心血管疾病相关的miRNA,经qPCR对其进行验证,通过生物信息学分析预测其调控靶基因;合成miRNA21 mimics转染CMVECs细胞,同时合成miRNA21 inhibitor,与CVB3共转染CMVECs细胞,检测各组细胞Caspase-3活性变化和细胞凋亡。结果 CVB3感染CMVECs细胞48 h后与正常对照组比较Caspase-3活性显著上调(P<0.01),细胞凋亡明显增加;通过基因芯片检测及生物信息学分析后发现,与心血管系统疾病密切相关的miRNA为miRNA21,经qPCR验证结果一致,预测其靶基因为PDCD4;miRNA21mimics转染CMVECs细胞后与正常对照组相比Caspase-3活性显著上调,细胞凋亡增加(P<0.05);而miRNA21 inhibitor与CVB3共转染CMVECs细胞后与CVB3感染组相比Caspase-3活性显著下调,细胞凋亡明显减少(P<0.05)。结论 miRNA21在CVB3感染的CMVECs细胞中的表达有显著变化,并与CMVECs细胞凋亡密切相关,提示miRNA21在CVB3诱导的病毒性心肌炎发病过程中可能起着重要作用。Objective In the study, we cultured cardiac microvascular endothelial cells （CMVECs） in vitro and infected CMVECs with CVB3 to scan miRNA by miRNAs oligonucleotide microarray technology. Moreover, we explored the effects of miRNA on CVB3-induced CMVECs apoptosis. Methods We cultured primary rat CMVECs and assessed activity of easpase-3 in CMVECs exposure to CVB3 with 48 hours. RNA was isolated and scanned by miRNAs oligonucleotide microarray. We choosed the miRNA which was expressed significantly different and closely related with cardiovascular diseases, and furtherly validated by qPCR. Additionally, we respectively used miKNA mimics and miRNA inhibitors to transfect CMVEC and detect caspases-3 activity. Results CVB3 significantly up-regulated caspase-3 activity in 48 hours, as compared with control （P〈0.05）. miRNA21 was increased by CVB3 and displayed close relation with cardiovascular diseases. Similarly, qPCR also showed miRNA21 levels were significantly raised. The activity of easpase-3 was elevated in miKNA21 mimics＋CVB3 group, which was decreased in miRNA21 inhibitors＋CVB3 group. Conclusion Our investigations showed that the expression of miRNA21 was markedly increased by CVB3 and miRNA21 regulated CMVECs apoptosis,suggesting miR.NA21 had vital roles in CVB3-induced viral myooarditis.

关 键 词：柯萨奇B3病毒 心脏微血管内皮细胞 miRNA21 

分 类 号：R542.21[医药卫生—心血管疾病]