微小核糖核酸-149-5p对人瓣膜间质细胞成骨样分化影响的研究  

Effects of miR-149-5p on the osteogenic differentiation of human aortic valve interstitial cells

作　　者：张帆[1] 姚可欣 张燕红 洪诗瑶 靳姝慧 张聪聪[1] ZHANG Fan;YAO Kexin;ZHANG Yanhong;HONG Shiyao;JIN Shuhui;ZHANG Congcong(Beijing Anzhen Hospital,Capital Medical University/Beijing Institute of Heart,Lung and Vascular Diseases,Beijing 100029,China)

机构地区：[1]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所,100029

出　　处：《心肺血管病杂志》2024年第5期486-492,共7页Journal of Cardiovascular and Pulmonary Diseases

基　　金：北京市自然科学基金(Z200026,7222048)。

摘　　要：目的:观察微小RNA(miR)-149-5p对瓣膜钙化细胞模型的影响,探讨其可能的机制。方法:收集正常瓣膜组织和钙化瓣膜组织并提取RNA,检测miR-149-5p及钙化相关基因Runt及转录因子2(Runt related transcription factor 2,RUNX2)的表达水平,并分析相关性;培养原代人瓣膜间质细胞(human valve interstitial cells,hVICs),更换成骨样分化诱导培养基(osteogenic differentiation medium,OM)培养14天,茜红素染色检测细胞成骨样分化程度,realtime-PCR检测miR-149-5p表达水平;进而将hVICs分为三组:对照组,NC+OM组和miR-149-5p+OM组,后两组分别转染NC mimic和miR-149-5p mimic;realtime-PCR检测细胞中成骨样分化标志基因OCN、ALPP和靶基因的mRNA表达水平,TargetScan和miRDB数据库预测miR-149-5p的靶基因,双荧光素酶报告基因实验验证miR-149-5p对靶基因的靶向关系。结果:与无钙化的相比,钙化的主动脉瓣膜组织中miR-149-5p的表达量显著下降,而且与RUNX2的表达量成负相关(均为P<0.05);hVICs诱导成骨样分化后茜红素染色阳性区域显著增加,同时miR-149-5p的表达逐渐下调(P<0.05);miR-149-5p mimic转染hVICs后,miR-149-5p+OM组细胞内miR-149-5p的表达较NC+OM组显著上调。miR-149-5p+OM组的hVICs中钙含量、OCN和ALPP的表达均低于NC+OM组(P<0.05)。TargetScan数据库预测结果显示miR-149-5p可与促成骨样分化的基因IL6的3’-UTR区结合;双荧光素酶报告基因实验显示miR-149-5p可抑制IL6报告基因活性(P<0.05),而报告基因突变后抑制作用消失;miR-149-5p+OM组hVICs中IL6的表达显著下调(P<0.05)。结论:miR-149-5p可能通过靶向抑制促钙化因子IL-6的表达,在主动脉瓣膜钙化中发挥保护性作用。Objective:To investigate the effects of the miR-149-5p on osteogenic differentiation of aortic valve interstitial cells(VICs)and explore its possible mechanism.Methods:Normal and calcified aortic valve tissues were collected,sample RNAs were extracted to detect the expression level of miR-149-5p,and correlation analysis was performed with the expression levels of miR-149-5p and calcification related genes Runt related transcription factor 2(RUNX2).Human VICs were cultured and induced by osteogenic differentiation medium(OM).Alizarin staining was used to detect the degree of osteogenic differentiation,and real-time PCR was used to detect the expression levels of miR-149-5p.VICs were divided into three groups:control group,NC+OM group,and miR-149-5p+OM group.The latter two groups were transfected with NC mimic and miR-149-5p mimic,respectively.Real-time PCR was used to detect the effect of miR-149-5p overexpression on the expression of osteogenic genes and target genes.TargetScan and miRDB database were used to predict the target genes of miR-149-5p,dual luciferase reporter system was used to detect the luciferase activity of target genes 3’-UTR,and Results:The expression of miR-149-5p was decreased in calcified aortic valves and was negatively correlated with the expression of RUNX2(P<0.05).After osteogenic differentiation induction,the positive area of Alizarin staining in human VICs was increased,while the expression of miR-149-5p decreased(P<0.05).After human VICs were transfected with miR-149-5p mimic,the expression of miR-149-5p in the cells was significantly up-regulated compared with NC+OM group(P<0.05).At the same time,the calcium content and the expression of OCN and ALPP in human VICs were lower in the miR-149-5p+OM group than that in the NC+OM group(P<0.05).Pro-osteogenic gene IL6 was predicted as the target genes of miR-149-5p by TargetScan database.miR-149-5p could inhibit the luciferase activity of IL63’-UTR(P<0.05).Real-time PCR results showed that the expression of IL6 in the hVICs of miR-149-5

关 键 词：主动脉瓣钙化 瓣膜间质细胞 成骨样分化 微小核糖核酸 

分 类 号：R54[医药卫生—心血管疾病]