MicroRNA-486 和TGF-β1 对人主动脉瓣膜 钙化的影响及其机制研究

Effect of microRNA-486 and TGF- β1 on calcification of human aortic valve and its mechanism

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-486 mimic、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-β1（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-β1、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-β1 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。

Abstract:

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.