Zhang Y, Sun L, Sun H, Liu X, Luo X, Li C, Sun D, Li T
Wuhan Children's Hospital (Wuhan Maternal and Child Healthcare Hospital), Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430000, P.R. China.
Taihe Hospital, Hubei University of Medicine, Shiyan 442000, P.R. China.
Cell Mol Biol (Noisy-le-grand). 2017 Oct 31;63(10):80-86. doi: 10.14715/cmb/2017.63.10.13.
The present study is to measure the expression of microRNA (miRNA or miR)-133b in circulating blood of children with viral myocarditis before and after drug treatment, and to investigate its relationship with the severity of myocardial lesions. A total of 36 children patients with viral myocarditis who received treatments at our hospital between June 2014 and June 2016 were enrolled in the present study, including 21 boys and 15 girls (age range, 9 months - 16 years).Quantitative real-time polymerase chain reaction was used to determine the expression of miR-133b in peripheral blood of patients and cardiomyocytes infected with CVB3. CCK-8 assay was used to test the proliferation of cardiomyocytes. ELISA was used to determine the levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) in peripheral blood and cardiomyocyte culture supernatants. Western blotting and ELISA were performed to measure the levels of tumor necrosis factor-α and interleukin-6 in cardiomyocytes infected by CVB3 and cell culture supernatants. Bioinformatics was used to predict the target gene of miR-133b. Silencing of Rab27B gene was achieved by transfection with its small-interfering RNA. Dual luciferase reporter assay was carried out to test whether miR-133b directly targets Rab27B. Reduced expression of miR-133b in peripheral blood was possibly correlated with myocardial injuries in viral myocarditis miR-133b. Expression of miR-133b was significantly reduced in cardiomyocytes infected with CVB3 virus. Overexpression of miR-133b inhibited cardiomyocyte injuries caused by CVB3 virus infection, and the enhanced production and release of cytokines TNF-α and IL-6 by cardiomyocytes infected with CVB3 virus. Rab27B promoted injuries of cardiomyocytes induced by CVB3 infection and facilitated the synthesis and release of cytokines TNF-α and IL-6 by cardiomyocytes. miR-133b was able to bind to the 3'-untranslated region seeding region of Rab27B mRNA. The present study demonstrates that expression of miR-133b in peripheral blood from children with viral myocarditis is reduced, and negatively correlated with myocardial injuries. miR-133b inhibits the proliferation of cardiomyocytes and the release of cytokines TNF-α and IL-6, and alleviates CVB3 infection-induced myocardial injuries by targeting Rab27B.
本研究旨在检测药物治疗前后病毒性心肌炎患儿循环血液中微小RNA（miRNA或miR）-133b的表达情况，并探讨其与心肌损伤严重程度的关系。本研究纳入了2014年6月至2016年6月期间在我院接受治疗的36例病毒性心肌炎患儿，其中男21例，女15例（年龄范围为9个月至16岁）。采用定量实时聚合酶链反应测定患者外周血及感染柯萨奇病毒B3（CVB3）的心肌细胞中miR-133b的表达。采用CCK-8法检测心肌细胞的增殖情况。采用酶联免疫吸附测定法（ELISA）测定外周血及心肌细胞培养上清液中肌酸激酶（CK-MB）和乳酸脱氢酶（LDH）的水平。采用蛋白质免疫印迹法和ELISA检测CVB3感染的心肌细胞及细胞培养上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的水平。利用生物信息学预测miR-133b的靶基因。通过转染小干扰RNA实现Rab27B基因的沉默。进行双荧光素酶报告基因测定以检测miR-133b是否直接靶向Rab27B。外周血中miR-133b表达降低可能与病毒性心肌炎中的心肌损伤相关。在感染CVB3病毒的心肌细胞中，miR-133b的表达显著降低。miR-133b过表达可抑制CVB3病毒感染引起的心肌细胞损伤，以及感染CVB3病毒的心肌细胞中细胞因子TNF-α和IL-6产生及释放的增加。Rab27B促进CVB3感染诱导的心肌细胞损伤，并促进心肌细胞中细胞因子TNF-α和IL-6的合成与释放。miR-133b能够与Rab27B mRNA的3'非翻译区种子序列结合。本研究表明，病毒性心肌炎患儿外周血中miR-133b表达降低，且与心肌损伤呈负相关。miR-133b通过靶向Rab27B抑制心肌细胞增殖及细胞因子TNF-α和IL-6的释放，并减轻CVB3感染诱导的心肌损伤。
