GLIS家族锌指蛋白2(GLIS2)负调控Wnt/β-catenin通路抑制人骨髓间充质干细胞的成骨分化

GLIS family zinc finger protein 2(GLIS2)negatively regulates the Wnt/β-catenin pathway to inhibit the osteogenic differentiation of human bone marrow mesenchymal stem cells

目的探究人类基因GLIS家族锌指蛋白2(GLIS2)对Wnt/β联蛋白(β-catenin)通路调控作用,及对人骨髓间充质干细胞(BMMSC)分化的影响。方法培养人BMMSC,随机分为空白组、成骨诱导组、腺病毒GLIS2基因过表达(ad-GLIS2)组、ad-GLIS2阴性对照、GLIS2基因敲低(si-GLIS2)组、si-GLIS2阴性对照(si-NC)组。反转录PCR检测GLIS2 mRNA表达判定转染情况,对硝基苯磷酸苯二钠(PNPP)法检测碱性磷酸酶(ALP)活性、茜素红染色法检测钙化结节形成判定其成骨特性;T细胞因子/淋巴增强因子(TCF/LEF)报告试剂盒检测细胞内Wnt/β-catenin通路激活情况;Western blot法检测GLIS2、Runt相关转录因子2(Runx2)、骨桥蛋白(OPN)、成骨细胞特异性转录因子(osterix)表达。谷胱甘肽巯基转移酶沉降(GST-pulldown)实验验证GLIS2与β-catenin二者相互作用关系。结果与空白组相比,成骨诱导组BMMSC的ALP活性及钙化结节形成增强,Wnt/β-catenin通路活性及成骨分化相关蛋白表达升高,成骨形成能力增强,GLIS2表达降低。上调GLIS2表达后,抑制BMMSC成骨分化能力,抑制Wnt/β-catenin通路活性及成骨分化相关蛋白表达,反之下调GLIS2表达后,可促进BMMSC成骨分化能力,提高Wnt/β-catenin通路活性及成骨分化相关蛋白表达。β-catenin与GLIS2之间有相互作用关系。结论GLIS2可能通过负调控Wnt/β-catenin通路活化,抑制BMMSC成骨分化能力。

Objective To investigate how the human GLIS family zinc finger protein 2(GLIS2)regulate the Wnt/β-catenin pathway and its influence on the differentiation of human bone marrow mesenchymal stem cells(BMMSCs).Methods Human BMMSCs were randomly divided into blank group,osteogenic induction group,GLIS2 gene overexpression(ad-GLIS2)group,ad-GLIS2 negative control group,gene knockdown(si-GLIS2)group,and si-GLIS2 negative control(si-NC)group.The expression of GLIS2 mRNA in each group was detected by reverse transcription-PCR to determine the transfection status;alkaline phosphatase(ALP)activity was detected by phenyl-p-nitrophenyl phosphate(PNPP),and calcified nodule formation was tested by alizarin red staining to determine its osteogenic properties;the activation of intracellular Wnt/β-catenin pathway was detected by T cell factor/lymphoid enhancer factor(TCF/LEF)reporter kit;the expression of GLIS2,Runt-related transcription factor 2(Runx2),osteopontin(OPN),and osterix was detected by Western blot analysis.The interaction between GLIS2 andβ-catenin was verified by GST-pulldown.Results Compared with the blank group,the ALP activity and calcified nodule formation of BMMSCs in the osteogenic induction group increased,and the Wnt/β-catenin pathway activity and the expression of osteogenic differentiation-related proteins increased,the osteogenic ability increased,while the expression of GLIS2 decreased.Up-regulating the expression of GLIS2 could inhibit the osteogenic differentiation of BMMSCs,while suppress the activity of the Wnt/β-catenin pathway and the expression of osteogenic differentiation-related proteins on the contrary.Down-regulating the expression of GLIS2 could promote the osteogenic differentiation of BMMSCs,and improve the activity of the Wnt/β-catenin pathway and the expression of osteogenic differentiation-related proteins.There was an interaction betweenβ-catenin and GLIS2.Conclusion GGLiS2may negatively regulate the activation of Wnt/β-catenin pathway and affect the osteogenic differentiation of BMMsCs.