MicroRNA-486和TGF-β_1对人主动脉瓣膜钙化的影响及其机制研究  

Effect of microRNA-486 and TGF-β_1 on calcification of human aortic valve and its mechanism

作　　者：蒋伟坚 方明 苏锦文 王温慧 王雪君 李新明[1] Wei-jian Jiang;Ming Fang;Jin-wen Su;Wen-hui Wang;Xue-jun Wang;Xin-ming Li(Zhoupu Hospital Affiliated to Shanghai University of Medicine&Health Sciences,Shanghai 201318,China)

机构地区：[1]上海健康医学院附属周浦医院,上海201318

出　　处：《中国现代医学杂志》2018年第32期25-32,共8页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:81560077);上海健康医学院种子基金(No:HMSF-16-22-030);上海市浦东新区卫计委优秀青年医学人才培养计划(No:PWRq2015-24)

摘　　要：目的研究microRNA-486(miRNA-486)对人主动脉瓣膜间质细胞(VICs)钙化的影响及作用机制。方法体外培养VICs,经成骨诱导后用双荧光素报告基因检测miRNA-486靶基因。转染miR-486mimic、miR-486 Inhibitors后,将细胞分为miR-486 mimic组、miR-486 mimic NC组、miR-486 Inhibitors组、miR-486 Inhibitors NC组。采用茜红素染色、ALP试验分析各组钙化程度,实时荧光定量聚合酶链反应(qRT-PCR)、免疫荧光试验检测miR-486靶基因及成骨相关因子mRNA和蛋白的表达水平。结果双荧光素报告基因检测和Western blot检测结果显示,转录生长因子-β_1(TGF-β_1)为潜在靶基因。茜红素染色、ALP试验结果显示,miR-486 mimic组钙化程度高于miR-486 Inhibitors组。qRT-PCR、免疫荧光试验结果表明,miR-486 mimic组TGF-β_1、SMAD3 mRNA和蛋白表达水平较低(P<0.05);miR-486 mimic组RUNX2、骨钙素mRNA表达水平较miR-486 Inhibitors组高(P<0.05)。结论 miR-486可通过下调TGF-β_1的表达,促进RUNX2、骨钙素的表达和活性,诱导间质细胞向成骨细胞分化。Objective To explore the effect of miRNA-486(miR-486)on calcification of human aortic valve interstitial cells(VICs)and its mechanism.Methods VICs were cultured in vitro.After osteogenesis induction the target genes of miR-486 were detected by dual luciferase report gene and Western blot.After transfection of miR-486 mimic and miR-486 inhibitors,VICs were divided into four groups including a miR-486 mimic group,a miR-486 mimic NC group,a miR-486 inhibitor group and a miR-486 inhibitor NC group.Alizarin red staining and ALP test were used to analyze the level of calcification.Meanwhile,qRT-PCR and immunofluorescence assay were applied to show the mRNA and protein expressions of miR-486 target gene and osteoblast-associated factors.Results The results of dual luciferase report gene and Western blot showed that the TGFB1 gene was the potential target gene and the results of alizarin red staining revealed that the degree of calcification in the miR-486 mimic group was higher than that in the miR-486 inhibitor group(P<0.05).Meanwhile,the mRNA and protein expression levels of the TGFB1 and SMAD3 decreased in the miR-486 mimic group(P<0.05);the mRNA levels of Runx2 and osteocalcin in the miR-486 mimic group were higher than those in the miR-486 inhibitor group(P<0.05).Conclusions miR-486 can promote the expression and activity of Runx2 and osteocalcin by down-regulating TGF-β1,and induce the differentiation of mesenchymal cells into osteoblasts.

关 键 词：心脏瓣膜疾病 microRNA-486 转录生长因子-β1 茜红素染色 碱性磷酸酶活性 主动脉瓣膜 

分 类 号：R542.5[医药卫生—心血管疾病]