炎症微环境下miR-30a调控Runx相关转录因子2对人牙周膜干细胞成骨分化的影响

Effect of miR-30a regulating Runx2 on the osteogenic differentiation of hPDLSCs in inflammatory microenvironment

目的:探究炎症微环境下微小RNA-30a(miR-30a)调控Runt相关转录因子2(Runx2)对人牙周膜干细胞成骨分化的影响。方法:体外培养人牙周膜干细胞(hPDLSCs),随机分为对照组、模型组、miR-30a mimics(模拟物)组、miR-30a mimics阴性对照组、miR-30a inhibitor(抑制剂)组、miR-30a inhibitor阴性对照组,除对照组外,其余各组以脂多糖诱导细胞炎症微环境,构建体外炎症模型,然后分组处理并诱导其成骨分化,采用碱性磷酸酶(ALP)及茜素红染色检测各组细胞成骨分化情况;采用酶联免疫吸附法(ELISA)检测各组细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平;采用实时荧光定量PCR(qRT-PCR)检测各组细胞miR-30a、成骨相关基因Runx2 mRNA表达水平;采用蛋白免疫印迹实验检测各组细胞Runx2、骨桥蛋白(OPN)、骨钙素(OCN)蛋白表达情况。结果:与对照组相比,模型组细胞miR-30a表达、细胞上清液中TNF-α及IL-6水平均升高(均P<0.05),Runx2 mRNA表达水平、ALP阳性细胞相对比例、矿化结节相对比例、Runx2、OPN及OCN蛋白表达水平均降低(均P<0.05)。与模型组相比,miR-30a mimics组细胞miR-30a表达、细胞上清液中TNF-α及IL-6水平均升高(均P<0.05),Runx2 mRNA表达水平、ALP阳性细胞相对比例、矿化结节相对比例、Runx2、OPN及OCN蛋白表达水平均降低(均P<0.05);miR-30a inhibitor组细胞miR-30a表达、细胞上清液中TNF-α及IL-6水平均降低(均P<0.05),Runx2 mRNA表达水平、ALP阳性细胞相对比例、矿化结节相对比例、Runx2、OPN及OCN蛋白表达水平均升高(均P<0.05);miR-30a mimics阴性对照组、miR-30a inhibitor阴性对照组细胞各指标无明显变化(均P>0.05)。结论:炎症微环境下hPDLSCs中miR-30a高表达,可下调Runx2表达,促进炎症进展,进而抑制hPDLSCs成骨分化。

Objective:To investigate the effect of microRNA-30a(miR-30a)regulating Runt-related transcription factor 2(Runx2)in inflammatory microenvironment on the osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs).Methods:The hPDLSCs were cultured in vitro and randomly divided into control group,model group,miR-30a mimics group,miR-30a mimics negative control group,miR-30a inhibitor group and miR-30a inhibitor negative control group.Except for the control group,the other groups used lipopolysaccharide to induce the inflammatory microenvironment of the cells to construct the inflammatory model in vitro,and then treated them in groups and induced their osteogenic differentiation.The alkaline phosphatase(ALP)and alizarin red staining were used to detect the osteogenic differentiation.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of TNF-αand IL-6 in the supernatant of each group.The expression levels of miR-30a,Runx2 mRNA were detected by qRT-PCR.The expression levels of Runx2,osteopontin(OPN)and osteocalcin(OCN)proteins were detected by Western blot.Results:Compared with the control group,the expression level of miR-30a and the levels of TNF-αand IL-6 in the supernatant of the model group were higher,the expression level of Runx2 mRNA,relative proportion of ALP positive cells,relative proportion of mineralized nodule,expression levels of Runx2,OPN and OCN proteins were lower(all P<0.05).Compared with the model group,the expression level of miR-30a and the levels of TNF-αand IL-6 in the supernatant of miR-30a mimics group were higher,the expression level of Runx2 mRNA,relative proportion of ALP positive cells,relative proportion of mineralized nodule,expression levels of Runx2,OPN and OCN proteins were lower(all P<0.05);the expression level of miR-30a and the levels of TNF-αand IL-6 in the supernatant of miR-30a inhibitor group were lower,the expression level of Runx2 mRNA,relative proportion of ALP positive cells,relative proportion of mineralized nodule,expression levels of Runx2,OPN and OCN proteins were higher(all P<0.05);there were no significant changes in cell indexes of miR-30a mimics negative control group and miR-30a inhibitor negative control group(all P>0.05).Conclusion:The miR-30a is highly expressed in hPDLSCs in inflammatory microenvironment,which can down-regulate the expression level of Runx2,promote the progress of inflammation,and then inhibit the osteogenic differentiation of hPDLSCs.