miRNA-132通过Wnt/β-catenin信号通路促进牙周膜干细胞成骨分化的研究

miRNA-132 Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells through Wnt/β-catenin Signaling Pathway

目的:探讨miRNA-132通过Wnt/β-catenin信号通路促进牙周膜干细胞的成骨分化。方法:细胞培养,观察牙周膜干细胞(periodontal ligament stem cells,PDLSCs)形态。成骨诱导,检测成骨样分化情况。成脂诱导,检测成脂分化情况。将PDLSCs细胞分为3组,分别为对照组、miRNA-132模拟组和miRNA-132抑制剂组,采用八肽胆囊收缩素(Cholecystokinin octapeptide,CCK-8)检测PDLSCs细胞增殖,酶联免疫检测碱性磷酸酶(Alkaline phosphatase protein,ALP)活性,茜素红染色检测矿化结节沉积,采用实时荧光定量PCR(Real-time quantitative PCR,qRT-PCR)法检测成骨相关基因,采用Western blot检测Wnt/β-catenin相关通路蛋白。结果:miRNA-132模拟组的PDLSCs细胞增殖能力、ALP活性及矿化结节数量显著高于对照组,miRNA-132抑制剂组明显低于对照组和miRNA-132模拟组,差异有统计学意义(P<0.05)。miRNA-132模拟组中的ALP、Runx2、OCN和Osterix mRNA的相对表达明显高于对照组,miRNA-132抑制剂组显著低于对照组和miRNA-132模拟组,差异均有统计学意义(P<0.05)。miRNA-132模拟组中的GSK3β和β-catenin蛋白的表达明显高于对照组,miRNA-132抑制剂组明显低于对照组和miRNA-132模拟组,差异有统计学意义(P<0.05)。XAV-939组中的ALP、Runx2、OCN和Osterix mRNA的相对表达明显低于对照组,miRNA-132模拟组表达高于对照组和XAV-939组,miRNA-132模拟组+XAV-939组表达高于XAV-939组,差异有统计学意义(P<0.05)。结论:过表达miRNA-132可以促进牙周膜干细胞的成骨分化,抑制miRNA-132表达从而抑制了人牙周膜干细胞的成骨分化,miRNA-132调节牙周膜干细胞的成骨分化,其机制可能是通过Wnt/β-catenin信号通路来实现的。

Objective To investigate the effect of miRNA-132 on osteogenic differentiation of periodontal ligament stem cells through Wnt/β-catenin signaling pathway.Methods The morphology of PDLSCs was observed by cell culture.Osteogenic induction and osteogenic differentiation were detected.Adipogenic induction and adipogenic differentiation were detected.PDLSCs cells were divided into three groups:the control group,the miRNA-132 simulation group and the miRNA-132 inhibitor group.The proliferation of PDLSCs cells was detected by CCK-8.ALP activity was detected by enzyme-linked immunosorbent assay.Mineralized nodule deposition was detected by alizarin red staining.Osteogenesis related genes were detected by qRT-PCR,and Wnt/β-catenin related pathway protein was detected by Western blot.Results TThe proliferation ability,ALP activity and the number of mineralized nodules of PDLSCs in the miRNA-132 simulation group were significantly higher than those in the control group,the differences were statistically significant(P<0.05).The miRNA-132 inhibitor group was significantly lower than the control group and miRNA-132 simulation group(P<0.05).The relative expression of ALP,Runx2,OCN and Osterix mRNA in the miRNA-132-132 simulation group was significantly higher than those in the control group,and the miRNA-132 inhibitor group was significantly lower than the control group and miRNA-132 simulation group(P<0.05).The expression of GSK3βandβ-catenin protein in the miRNA-132 simulation group was significantly higher those that in control group,and the miRNA-132 inhibitor group was significantly lower than the control group and miRNA-132 simulation group(P<0.05).The relative expression of ALP,Runx2,OCN and Osterix mRNA in the XAV-939 group was significantly lower than those in the control group(P<0.05).The expression in the miRNA-132 simulation group was higher than those in the control group and the XAV-939 group(P<0.05).The expression in the miRNA-132 simulation+XAV-939 group was higher than those in the XAV-939 group(P<0.05).Conclusion Overexpression of miRNA-132 can promote the osteogenic differentiation of periodontal ligament stem cells,and inhibit the expression of miRNA-132,thus inhibiting the osteogenic differentiation of human periodontal ligament stem cells.The mechanism of miRNA-132 regulating the osteogenic differentiation of periodontal ligament stem cells may be through Wnt/β-catenin signaling pathway.