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目的:研究分化抑制因子1(inhibitor of differentiation 1,Id1)基因对人骨肉瘤细胞MG63凋亡的影响,并探讨可能的作用机制。方法 :通过重组腺病毒感染的方法将特异性针对Id1基因的Id1-si RNA转入骨肉瘤细胞MG63中,分别采用半定量RT-PCR和蛋白质印迹法检测Id1 m RNA和蛋白的表达水平;AnnexinⅤ-FITC单染法及DAPI染色法检测对细胞凋亡的影响,FCM法检测对细胞周期分布的影响。蛋白质印迹法检测凋亡相关蛋白Bcl-2和survivin,以及Wnt信号通路关键蛋白β-链蛋白(β-catenin)、受体酪氨酸激酶样孤儿受体(2receptor tyrosine kinase-like orphan receptor 2,ROR2)和CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)蛋白的表达水平。结果:采用重组腺病毒Ad-Id1-siR NA感染MG63细胞后,Id1 mR NA及蛋白的表达水平均明显下调(P值均<0.05);AnnexinⅤ-FITC单染法和DAPI染色检测结果显示,沉默Id1基因表达后MG63细胞的凋亡率明显提高(P值均<0.05);半定量RT-PCR和蛋白质印迹法检测结果显示,抗凋亡因子Bcl-2和survivin mR NA及蛋白的表达水平均明显下调(P值均<0.05);蛋白质印迹法检测结果显示,Wnt信号通路关键蛋白β-catenin以及Wnt通路相关蛋白ROR2和CHOP的表达水平均明显下调(P值均<0.05)。结论 :靶向沉默Id1基因表达能促进骨肉瘤细胞的凋亡,其作用可能与抑制Wnt信号通路的活性有关。
Objective: To investigate the effect of Id1 gene on the apoptosis of human osteosarcoma MG63 cells and to explore the possible mechanism. Methods: The Id1-si RNA specific to Id1 gene was transfected into MG63 cells by recombinant adenovirus infection. The expression of Id1 m RNA and protein was detected by semi-quantitative RT-PCR and Western blot respectively. AnnexinⅤ The effects of FITC staining and DAPI staining on cell apoptosis were detected by FCM and the cell cycle distribution was analyzed by FCM. Western blotting was used to detect the expression of apoptosis-related proteins Bcl-2 and survivin, as well as β-catenin, Wnt signaling pathway key receptor 2 receptor tyrosine kinase-like orphan receptor 2 ROR2) and CCAAT / enhancer-binding protein homologous protein (CHOP). Results: The expression of Id1 mRNA and protein in MG63 cells was significantly downregulated by recombinant adenovirus Ad-Id1-siR NA (all P <0.05). The result of Annexin Ⅴ-FITC staining and DAPI staining showed that silencing The apoptosis rate of MG63 cells was significantly increased after Id1 gene expression (all P <0.05). The results of semi-quantitative RT-PCR and Western blot showed that the expressions of anti-apoptotic factor Bcl-2, survivin mR NA and protein (P <0.05). The results of Western blotting showed that the expressions of key proteins of Wnt signaling pathway such as β-catenin and Wnt pathway related proteins ROR2 and CHOP were significantly down-regulated (all P <0.05). Conclusion: Targeted silencing Id1 gene expression can promote the apoptosis of osteosarcoma cells, which may be related to the inhibition of Wnt signaling pathway.