miR-155在炎症微环境下对牙周膜干细胞成骨分化的调控作用

Regulation effect of miR-155 on osteogenic differentiation of periodontal ligament stem cells in inflammatory microenvironment

目的 研究miR-155在炎症微环境下对牙周膜干细胞(periodontal ligment stem cells, PDLSCs)成骨分化的调控作用。方法 将PDLSCs按照不同处理方法分为对照组、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)组、miR-阴性对照(NC)组、TNF-α+miR-NC组、TNF-α+miR-155抑制物组、miR-155组,采用荧光定量PCR检测miR-155、Runt相关转录因子2(recombinant runt related transcription factor 2,Runx2)、骨钙素(osteocalcin, OCN)、Ⅰ型胶原(collagenⅠ,Col-Ⅰ)、骨形态发生蛋白5(bone morphogenetic protein 5,BMP5)、骨形态发生蛋白10(bone morphogenetic protein 10,BMP10) mRNA表达水平;Western blot检测Runx2、OCN、Col-Ⅰ、BMP5、BMP10蛋白表达;茜素红染色检测细胞钙结节形成;双荧光素酶报告基因验证miR-155靶向BMP5、BMP10。结果 与对照组比较,TNF-α组PDLSCs中miR-155的表达水平明显升高,Runx2、OCN、Col-Ⅰ的mRNA和蛋白表达水平以及钙结节形成量降低(P<0.05);与miR-NC组比较,TNF-α+miR-NC组PDLSCs中Runx2、OCN、Col-Ⅰ的mRNA和蛋白表达水平及钙结节形成量降低,差异有统计学意义(P<0.05);与TNF-α+miR-NC组比较,TNF-α+miR-155抑制物组PDLSCs中Runx2、OCN、Col-Ⅰ的mRNA和蛋白表达水平及钙结节形成量明显升高(P<0.05);miR-155靶向负调控BMP5、BMP10表达。结论 炎症微环境下PDLSCs中miR-155表达增加,敲低miR-155可促进炎症微环境下PDLSCs成骨分化,靶向调控BMP5及BMP10是相关分子机制。

Objective To study the regulatory effect of miR-155 on osteogenic differentiation of periodontal ligament stem cells(PDLSCs) in inflammatory microenvironment. Methods PDLSCs were cultured and divided into control group, tumor necrosis factor group-α(TNF-α) group, miR-negative control(NC) group, TNF-α+miR-NC group, TNF-α+miR-155 inhibitor group and miR-155 group according to different treatment methods. Fluorescence quantitative PCR was used to detect miR-155, recombinantrunt related transcription factor 2(Runx2), osteocalcin(OCN), collagen Ⅰ(Col-Ⅰ),bone morphogenetic protein 5(BMP5) and bone morphogenetic protein 10(BMP10) expression levels;Western blot was used to detect Runx2, OCN, Col-Ⅰ, BMP5, BMP10 protein expression;alizarin red staining was used to detect the formation of calcium nodules in cells;the double luciferase reporter gene was used to verify that miR-155 targeted BMP5 and BMP10. Results Compared with the control group, the expression level of miR-155 in PDLSCs in the TNF-α group increased;the mRNA and protein expression levels of Runx2, OCN, Col-Ⅰ and the formation of calcium nodules decreased(P<0.05). Compared with the miR-NC group, the mRNA and protein expression levels of Runx2, OCN and Col-Ⅰ and the formation of calcium nodules in PDLSCs in the TNF-α+miR-NC group were decreased(P<0.05). Compared with the TNF-α+miR-NC group, the mRNA and protein expression levels of Runx2, OCN and Col-Ⅰ and the formation of calcium nodules in PDLSCs in the TNF-α+miR-155 inhibitor group increased(P<0.05). miR-155 negatively regulated the expressions of BMP5 and BMP10. Conclusion The expression of miR-155 in PDLSCs increases in inflammatory microenvironment. Knocking down miR-155 promotes the osteogenic differentiation of PDLSCs in inflammatory microenvironment. Targeting BMP5 and BMP10 is the relevant molecular mechanism.