靶向抑制Id1基因调控Wnt信号通路促进骨肉瘤MG63细胞凋亡

Interference of Id1 gene expression promotes apoptosis of osteosarcoma MG63 cells by regulating Wnt signaling pathway

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 inhibitor of differentiation 1（Id 1） gene on apoptosis of human osteosarcoma MG63 cells, and to explore its possible molecular mechanism.Methods： The specific siR NA targeting Id 1 gene（Id1-siR NA） was infected into MG63 cells using recombinant adenovirus Ad-siR NA-Id1 as the AdsiI d1 group, while the negative control group（infection with the empty vector adenovirus AdR FP） and the blank control group（non-infection） were set up, respectively. The expression levels of Id1 mR NA and protein were detectedby semi-quantitative RT-PCR and Western blotting, respectively. The apoptosis of MG63 cells was detected by AnnexinⅤ-FITC staining and DAPI staining. The cell cycle distribution of MG63 cells was detected by FCM method. The expression levels of apoptosis-related proteins（Bcl-2 and survivin） and Wnt pathway-related proteins [β-catenin, receptor tyrosine kinase-like orphan receptor 2（ROR2） and CCAAT/enhancer-binding protein homologous protein（CHOP）] were detected by Western blotting. Results： After infection with recombinant adenovirus Ad-siR NA-Id1, the expression levels of Id1 mR NA and protein in MG63 cells were significantly decreased（both P〈 0.05）. The apoptotic rate in AdsiI d1 group was higher than those in AdR FP and the blank control groups（both P〈 0.05）. The expression levels of Bcl-2 and survivin mR NAs and proteins were significantly decreased（all P 〈0.05）. The expressions of β-catenin, ROR2 and CHOP proteins were also significantly decreased（all P 〈0.05）. Conclusion： The silencing of Id 1 gene expression can promote the apoptosis of osteosarcoma cells, which may be related to regulating Wnt signaling pathway.