DNA损伤检查点蛋白调节子1通过抑制p53通路促进胆管癌细胞的增殖、迁移及侵袭

MDC1 promotes proliferation,migration and invasion of cholangio-carcinoma cells by suppressing p53 signaling pathway

目的 探索DNA损伤检查点蛋白调节子1(MDC1)对胆管癌细胞增殖、迁移、侵袭、周期以及凋亡的影响及其机制。方法 采用特异性靶向MDC1的小干扰RNA(siRNA)在胆管癌细胞RBE和Huh28中瞬时敲低MDC1,使用pLVX-HA-MDC1质粒在RBE和Huh28中瞬时过表达MDC1;采用实时定量PCR(qPCR)和Western印迹鉴定敲低和过表达MDC1的效果。采用CCK-8、平板细胞克隆形成、Transwell和Invasion实验分别检测RBE和Huh28细胞的增殖、迁移和侵袭能力;采用流式细胞术检测细胞周期和凋亡;使用基因集富集分析(GSEA)预测与MDC1显著相关的信号通路,通过Western印迹验证通路相关节点分子的表达情况。采用免疫共沉淀(Co-IP)验证MDC1与p53的相互作用。采用流式细胞术和Western印迹验证MDC1是否通过p53通路影响RBE和Huh28的周期和凋亡。基于癌症基因组图谱(TCGA)数据库分析胆管癌癌组织与正常组织中MDC1的mRNA表达差异情况,以及MDC1表达水平与胆管癌患者总体生存率的相关性。结果 敲低MDC1,RBE和Huh28细胞的增殖、迁移、侵袭能力显著降低,S期细胞比例显著减少,G;0;/G;1;期细胞比例、凋亡率显著增加;而过表达MDC1,RBE和Huh28细胞的增殖、迁移及侵袭能力显著提高,S期细胞比例显著增多,G;0;/G;1;期细胞比例、凋亡率显著减少;在RBE和Huh28细胞中,证实MDC1与p53存在相互作用,且MDC1显著下调p53、p-p53(Ser-15)及p53通路下游分子B淋巴细胞瘤-2相关X蛋白(BAX)、p53上调凋亡调控因子(PUMA)、p21的表达,显著上调B淋巴细胞瘤-2(Bcl-2)的表达,从而促进胆管癌细胞的发生发展;MDC1在胆管癌组织中的表达水平显著高于正常胆管组织,且MDC1高表达与胆管癌患者预后不良密切相关。结论MDC1通过抑制p53通路促进胆管癌的发生发展,MDC1可作为新的胆管癌预后不良的标志物。

Objective To investigate the effect of the mediator of DNA damage check point protein 1(MDC1)on proliferation,migration,invasion,cell cycle and cell apoptosis in cholangiocarcinoma(CCA)and the potential molecular mechanism.Methods The small interfering RNA(siRNA)specifically targeting MDC1 was used to transiently knock down MDC1.Recombined plasmid containing MDC1 was transiently transfected into RBE and Huh28 cells for overexpression of MDC1.Real time quantitative PCR(qPCR)and Western blotting were adopted to verify the effectiveness of MDC1 knockdown or overexpression.The proliferation of CCA cells was measured via CCK-8 and cell colony formation assays.Transwell and Invasion assays were used to detect cell migration and invasion while flow cytometry assays were employed to detect cell cycle and apoptosis.Gene set enrichment analysis(GSEA)was conducted to investigate the pathways which were significantly associated with MDC1,and the expression of p53 downstream protein was verified by Western blotting assay.Co-immunoprecipitation(Co-IP)assays were used to verify the interactions between MDC1 and p53.Flow cytometry and Western blotting assays were performed to find out whether MDC1 promoted cell cycle and cell apoptosis through p53 pathway.Based on The Cancer Genome Altas(TCGA)database,the difference in MDC1 expression levels between CCA and normal tissues was analyzed,and the correlations between the MDC1 expression levels and the clinical prognosis of CCA patients were investigated.Results Knockdown of MDC1 in RBE and Huh28 cells significantly inhibited cells proliferation,migration and invasion,significantly decreased the proportion of cells in S phase,and significantly increased the proportion of cells in G0/G1 phase and apoptosis rate while overexpression of MDC1 could significantly promote cell proliferation,migration and invasion,significantly increase the proportion of cells in S phase,and significantly decrease the proportion of cells in G0/G1 phase and apoptosis rate.It was found that MDC1 interacted with p53 in RBE and Huh28 cells,and MDC1 significantly down-regulated the expressions of p53,p-p53(Ser-15),BAX,PUMA and p21,but significantly up-regulated the expression of Bcl-2,which in turn promoted the tumorigenesis of CCA.MDC1 was up-regulated in CCA tissues compared to the normal tissues,and the high expressions of MDC1 were significantly associated with poor clinical outcomes of CCA patients.Conclusion MDC1 promotes the development of CCA by suppressing the p53 pathway,and MDC1 may be a candidate marker for the poor prognosis in CCA.