miR-214-5p调控MFG-E8表达参与主动脉瓣膜间质细胞成骨样分化

miR-214-5p Affects Osteogenic Differentiation of Human Aortic Valvular Interstitial Cells by Regulating MFG-E8 Expression

目的研究乳脂球表皮生长因子8(milk fat globule-epidermal growth factor 8,MFG-E8)对主动脉瓣膜间质细胞(aortic valve interstitial cells,AVICs)成骨样分化的影响,并寻找调控其表达的微小RNA(microRNA,miRNA),探讨其在钙化性主动脉瓣疾病(calcific aortic valve disease,CAVD)发病机制中的作用。方法收集因CAVD行主动脉瓣置换手术患者的钙化瓣膜标本及因扩张型心肌病行心脏移植手术患者的非钙化瓣膜标本,通过免疫组织化学染色及Western blot实验检测MFG-E8在钙化瓣膜及非钙化瓣膜中的表达;分离AVICs进行体外培养,通过小干扰RNA(small interfering RNA,siRNA)沉默MFG-E8及成骨培养液(osteogenic medium,OM)干预,研究MFG-E8在AVICs成骨样分化中的作用;TargetScanHuman网站预测调控MFG-E8表达的miRNA并对其功能进行检测;real-time PCR检测miRNA在钙化及非钙化瓣膜中的表达差异。结果与非钙化瓣膜相比,钙化瓣膜中MFG-E8表达显著上调;以siRNA沉默MFG-E8显著抑制了OM诱导的AVICs成骨样分化;miR-214-5p可影响MFG-E8表达从而调控AVICs成骨样分化;相较于非钙化瓣膜,钙化瓣膜中miR-214-5p表达显著下调。结论miR-214-5p通过调控MFG-E8表达参与主动脉瓣膜间质细胞成骨样分化,在CAVD发病机制中可能发挥重要作用。

Objective To investigate the effect of milk fat globule-epidermal growth factor 8(MFG-E8)on the osteogenic differentiation of human aortic valvular interstitial cells and the pathogenesis of calcific aortic valve disease(CAVD),and to ex-plore the microRNA which can regulate the expression of MFG E8.Methods Calcified valve specimens were collected from CAVD patients underwent aortic valve replacement due to severe aortic stenosis,and non-calcified valve specimens were obtained from patients underwent heart transplant due to dilated cardiomyopathy.The expression levels of MFG E8 in calcified and non-calcified valves were detected by immunohistochemical staining and Western blotting.The role of MFG E8 in osteogenic differentiation of AVICs was studied by silencing MFG E8 with small interfering RNA(siRNA)and osteogenic medium(OM)treatment.The microRNAs regulating MFG E8 expression were predicted by TargetScanHuman website and their functions were detected.Finally,real-time PCR was used to detect the difference in microRNA expression between calcified and non-calci-fied valves.Results Compared with non-calcified valves,the expression of MFG E8 was significantly upregulated in calcified valves.Silencing MFG E8 by siRNA significantly inhibited OM induced osteogenic differentiation of AVICs.miR-214-5p could regulate the osteogenic differentiation of AVICs by interfering with the expression of MFG E8.Compared with non calcified valves,the expression of miR-214-5p was significantly downregulated in calcified valves.Conclusion miR-2145p is involved in osteogenic differentiation of AVICs by regulating MFG E8 expression,and plays an important role in the pathogenesis of CAVD.