miR-206通过IGF1对人牙周膜干细胞的成骨分化能力的影响及机制研究

Effects of miR-206 on the osteogenic differentiation property of human periodontal ligament stem cells through IGF1 and its underlying mechanism

目的:探讨miR-206影响人牙周膜干细胞(PDLSCs)成骨分化能力的分子机制。方法:分离培养PDLSCs并鉴定细胞表型,RT-qPCR检测成骨诱导前后细胞中miR-206表达水平;细胞随机分为对照组、miR-206 mimics组、mimics-NC组、miR-206 inhibitor组和inhibitor-NC组及miR-206 mimics+NC组和miR-206 mimics+胰岛素样生长因子-1(IGF-1)组,MTT法检测细胞增殖;碱性磷酸酶(ALP)染色检测细胞ALP活性;茜素红染色检测细胞矿化能力;RT-qPCR检测细胞中miR-206和IGF-1 mRNA水平;Western blot检测细胞中Runt相关转录因子2(RUNX2)、骨桥蛋白(OPN)和ALP蛋白表达水平;双荧光素酶报告基因系统检测miR-206与IGF-1的靶向性。结果:成骨诱导后PDLSCs中miR-206表达明显低于诱导前(P<0.05)。与mimics-NC组比较,miR-206 mimics组PDLSCs增殖能力、ALP活性、矿化能力、RUNX2、OPN和ALP蛋白表达显著下降(P<0.05),miR-206 inhibitor组上述指标趋势相反(P<0.05)。与miR-206 mimics+NC组比较,miR-206 mimics+IGF-1组ALP活性、矿化能力、RUNX2、OPN和ALP蛋白表达显著升高(P<0.05)。双荧光素酶实验结果显示,IGF-1是miR-206的靶基因。结论:miR-206可抑制PDLSCs增殖和成骨分化,可能与靶向抑制IGF-1表达有关。

Objective:To explore the molecular mechanism of miR-206 affecting the osteogenic differentiation property of human periodontal ligament stem cells(PDLSCs).Methods:PDLSCs were isolated and cultured,and the cell phenotypes were identified.miR-206 expression in cells before and after osteogenic induction were detected by RT-qPCR.The cells were randomly divided into control group,miR-206 mimics group,mimics-NC group,miR-206 inhibitor group,inhibitor-NC group and miR-206 mimics+NC group and miR-206 mimics+insulin-like growth factors-1(IGF-1)group,MTT was used to detect cell proliferation,alkaline phosphatase(ALP)staining was performed to analyze cell ALP activity,alizarin red staining was used to detect cell mineralization ability,the miR-206 and IGF-1 mRNA,the Runt-related transcription factor 2(RUNX2),osteopontin(OPN)and ALP protein expression levels in cells were detected by RT-qPCR and western blot respectively.Dual luciferase reporter gene system was used to detect the targeting relationship of miR-206 and IGF-1.Results:The expression of miR-206 in PDLSCs after osteogenic induction was significantly lower than that before induction(P<0.05).Compared with the mimics-NC group,the proliferation,ALP activity,mineralizationas well as the expression of RUNX2,OPN and ALP protein in PDLSCs in the miR-206 mimics group were significantly decreased(P<0.05),and the trend of the above indicators in the miR-206 inhibitor group was opposite(P<0.05).Compared with the miR-206 mimics+NC group,the ALP activity,mineralization ability,RUNX2,OPN and ALP protein expression in the miR-206 mimics+IGF-1 group were significantly increased(P<0.05).The results of dual luciferase experiments showed that IGF-1was the target gene of miR-206.Conclusion:miR-206 could inhibit the proliferation and osteogenic differentiation of PDLSCs,which may be related to the targeted inhibition of IGF-1 expression.