miR-203靶向RUNX2对牙周膜干细胞成骨分化能力的调控作用

Study on effect of miR-203 for targeting RUNX2 on osteogenic differentiation of human periodontal ligament stem cells

目的:体外研究miR-203对人牙周膜干细胞成骨分化能力的影响及调控机制。方法:分离培养人牙周膜干细胞,检测牙周膜干细胞矿化诱导前后miR-203和RUNX2的表达;分别转染miR-203沉默及过表达模拟物;qPCR检测miR-203和RUNX2 mRNA水平的表达;Western blot检测RUNX2蛋白水平的表达,茜素红染色观察细胞成骨分化能力的变化;生物信息学分析miR-203的靶基因,双荧光素酶报告实验验证;共转染miR-203 inhibitor和RUNX2 siRNA,分析共转染后对RUNX2表达及细胞成骨分化能力的影响。结果:成骨诱导液诱导后牙周膜干细胞中miR-203的表达水平显著下调,而RUNX2的表达水平明显升高;miR-203过表达可明显抑制h PDLSCs的成骨分化能力和RUNX2的表达,抑制miR-203的表达,则结果相反;通过生物信息学分析miR-203的潜在靶基因含有RUNX2,双荧光素酶报告实验验证RUNX2为miR-203的靶基因;进一步实验结果表明沉默RUNX2可逆转miR-203 inhibitor对牙周膜干细胞成骨分化的促进作用。结论:miR-203可靶向调控RUNX2对人牙周膜干细胞成骨分化能力产生影响,提示miR-203在h PDLSCs组织工程损伤修复过程中发挥着重要作用。

Objective: To investigate regulation of miR-203 on the osteogenic differentiation capacity of human periodontal ligamentstem cell(hPDLSCs) in vitro. Methods: The expression of miR-203 and RUNX2 were detected in h PDLSCs under conduction of bone induction for two weeks. Periodontal ligament stem cells were isolated from human healthy periodontal ligament tissue and infected with miR-203. Then the osteogenic differentiation capacity were evaluated alizarin red assay. The expression of miR-203 and RUNX2 were detected by q-RCR and Western blot. The potential target genes of miR-203 were analyzed by bioinformatics and verified by dual luciferase report. Results: The expression of miR-203 were significant down-regulated but RUNX2 were up-regulated under the conduction of bone induction. The osteogenic differentiation capacity of h PDLSCs were inhibited after overexpression of miR-203. RUNX2 was the potential target gene of miR-203, which verified by bioinformatics analysis and dual luciferase report. In addition, si-RUNX2 was able to reverse the induced effect of miR-203 inhibitor on the osteogenic differentiation of hPDLSCs. Conclusion: miR-203 inhibit the osteogenic differentiation ability of hPDLSCs via inhibiting the expression of RUNX2, it was suggested that miR-203 plays an important role in hPDLSCs involved in injury repair.