miR-214抑制牙囊细胞成骨分化的体外研究

mi R-214 inhibits the osteogenic differentiation of dental follicle cells in vitro

目的探讨miR-214对于牙囊细胞(dental follicle cells,DFCs)成骨分化的影响。方法双向差速传代法体外培养提纯牙囊细胞,以未转染的DFCs为对照(DFCs组),通过向DFCs中转染miR-214-3p mimics(miR-214 mimics组)或miR-214-3p inhibitor(miR-214 inhibitor组)分别上调和下调DFCs中的miR-214的表达。成骨诱导7 d,qRT-PCR检测miR-214表达及相关成骨基因碱性磷酸酶(alkaline phosphatase,ALP)、骨粘连蛋白(osteonectin,OSN)、成骨相关转录因子2(runt-related transcription factor-2,RUNX-2)的mRNA表达,免疫蛋白印迹检测各组RUNX-2、β-catenin蛋白表达;成骨诱导14 d茜素红染色观察矿化结节形成的情况。结果骨诱导7d,相较DFCs组,miR-214 mimics组miR-214的表达上调,miR-214 mimics组成骨相关基因ALP、OSN及RUNX-2的mRNA表达均低于DFCs组,但仅ALP在两组的差异具有统计学意义(P>0.05),而miR-214 inhibitor组成骨相关基因ALP、OSN、RUNX-2的mRNA表达均高于DFCs组,且差异均具有统计学意义(P<0.05)。miR-214mimics组RUNX-2、β-catenin的蛋白表达均低于miR-214 inhibitor组。miR-214 mimics组钙化结节明显少于DFCs组,而miR-214 inhibitor组钙化结节却明显多于DFCs组。结论miR-214上调可使β-catenin表达下调,并抑制成骨相关基因ALP、OSN及RUNX-2的表达,抑制成骨分化;下调miR-214,结果相反;miR-214可能是通过下调β-catenin的表达,抑制DFCs的成骨分化。

Objective To investigate the effect of miR-214 on the osteogenic differentiation of dental follicle cells(DFCs).Methods Purified DFCs were cultured in vitro by bidirectional differential passage,with the untransfected DFCs as the control group(DFCs group).The expression of miR-214-3p in DFCs was upregulated and downregulated by transfection of miR-214-3p(miR-214 mimics group)or miR-214-3p inhibitors(miR-214 inhibitor group)into DFCs.The expression levels of miR-214,alkaline phosphatase(ALP),osteonectin(OSN)and runt-related transcription factor-2(RUNX-2)were detected by qRT-PCR after 7 days of osteogenesis induction,the protein expression levels of RUNX-2 andβ-catenin were detected by western blot,and the formation of mineralized nodules was observed with alizarin red staining after 14 days of osteogenesis induction.Results Compared with the DFCs group,in the miR-214 mimics group,the expression of miR-214 was upregulated after 7 days of osteogenesis induction.The mRNA expression of ALP,OSN and RUNX-2 in the miR-214 mimics group was lower than that in the DFCs group,but only ALP in the two groups was statistically significant(P>0.05);the mRNA expression of ALP,OSN and RUNX-2 in the miR-214 inhibitor group was higher than that in the DFCs group,and the difference was statistically significant(P<0.05).The protein expression of RUNX-2 andβ-catenin in the miR-214 mimics group was lower than that in the miR-214 inhibitor group.The number of calcified nodules in the miR-214 mimics group was significantly less than that in the DFCs group,while that in the miR-214 inhibitor group was significantly higher than that in the DFCs group.Conclusion The upregulation of miR-214 can downregulate the expression ofβ-catenin,can inhibit the expression of ALP,OSN and RUNX-2 related to osteogenesis,and can inhibit osteogenic differentiation.The downregulation of miR-214 demonstrated the opposite results;miR-214 may downregulate the expression ofβ-catenin and inhibit the osteogenic differentiation of DFCs.