miR-27a-3p靶向调控Yes相关蛋白1促进人牙髓干细胞成骨分化

Effect of miR-27a-3p on osteoblast differentiation of human dental pulp stem cells via targeting YAP1

目的探讨miR-27a-3p靶向调控Yes相关蛋白1(YAP1)基因对人牙髓干细胞(hDPSCs)成骨分化的影响。方法体外分离培养hDPSCs细胞,将miR-27a-3p模拟物(miR-27a-3p mimics,过表达组)、miR-27a-3p抑制剂(miR-27a-3p inhibitor,敲低组)、miR-NC(对照组)分别转染至hDPSCs细胞中,检测miR-27a-3p表达水平的改变对YAP1表达的影响。采用TargetScan数据库检索miR-27a-3p的靶基因。应用双荧光素酶报告基因实验验证miR-27a-3p和YAP1的靶向关系。将miR-27a-3p mimics、miR-NC转染到hDPSCs特定时间后收集细胞,应用实时荧光定量PCR(qRT-PCR)、Western blot等检测经典的成骨标志物骨形态发生蛋白-2(BMP-2)、成骨转录因子-2(RUNX2)、骨桥蛋白(OPN)、YAP1 mRNA和蛋白表达水平。应用MTT比色法验证miR-27a-3p和YAP1对细胞增殖活性的影响。结果过表达组hDPSCs细胞中miR-27a-3p的表达水平较对照组显著上调(P<0.05),YAP1 mRNA和蛋白的表达水平下调(P<0.05)。而敲低组miR-27a-3p的表达水平较对照组明显下降(P<0.05),YAP1 mRNA和蛋白的表达水平增高(P<0.05)。生物信息学分析表明,miR-27a-3p作用于YAP1的3′UTR区,双荧光素酶报告基因实验验证了YAP1是miR-27a-3p的靶基因。qRT-PCR、Western blot检测显示,与对照组相比,miR-27a-3p过表达时hDPSCs中BMP-2、RUNX2、OPN mRNA和蛋白的表达水平上调(P<0.05)。MTT检测显示,miR-27a-3p过表达可明显上调hDPSCs增殖率(P<0.05),而YAP1过表达则会降低hDPSCs的增殖率(P<0.05)。结论miR-27a-3p可能通过靶向调控YAP1促进hDPSCs成骨分化。

Objective To explore the function of miR-27a-3p on osteoblast differentiation of hDPSCs by targeting Yes-related protein 1(YAP1).Methods Human dental pulp stem cells(hDPSCs)were cultured in vitro and transfected with miR-27a-3p mimics(miR-27a-3p overexpression group),miR-27a-3p inhibitor(miR-27a-3p knock-down group)and miR-NC(control group),respectively.Real-time PCR(qRT-PCR)and Western blot were used to detect the effect of miR-27a-3p expression levels on YAP1.Bioinformatics predicted the target gene of miR-27a-3p which was then verified by dual luciferase reporter gene experiments.In addition,miR-27a-3p mimics and miR-NC were transfected into hDPSCs at the time stated.The expression of miR-27a-3p and the biochemical markers of bone formation of hDPSCs,such as bone morphogenetic protein-2(BMP-2)and runt-related transcription factor 2(RUNX2)and osteopontin(OPN),YAP1 mRNA was detected by qRT-PCR.Western blot was applied to detect the expression of BMP-2,RUNX2,OPN and YAP1 protein.The effect of miR-27a-3p or YAP1 on the proliferation of hDPSCs was analyzed by MTT assay.Results The expression of miR-27a-3p in the miR-27a-3p overexpression group increased in comparison with the control group(P<0.05),when that of YAP1 mRNA and protein was suppressed(P<0.05).Compared with control group,the expression of miR-27a-3p in miR-27a-3p knock-down group decreased(P<0.05)as that of YAP1 mRNA and protein increased(P<0.05).The target gene of miR-27a-3p was YAP1,which was predicted by bioinformatics and verified by dual luciferase reporter gene experiments(P<0.05).Overexpression of miR-27a-3p could promote expression of BMP-2,RUNX2,OPN mRNA and protein(P<0.05)while down-regulating the expression of YAP1 mRNA and protein.Overexpression of YAP1 could mitigate the effect of miR-27a-3p on proliferation of hDPSCs(P<0.05).Conclusion miR-27a-3p promotes osteogenic differentiation of hDPSCs,which may be related to the down-regulation of YAP1 by miR-27a-3p.