ANLN在肝内胆管癌中的表达及对胆管癌细胞增殖的影响

Expression of ANLN in intrahepatic cholangiocarcinoma and its effect on proliferation of cholangiocarcinoma cells

目的:探讨ANLN基因在肝内胆管癌组织中的表达情况以及在胆管癌细胞增殖中的作用及机制。方法:收集手术切除的40例肝内胆管癌患者癌组织及癌旁组织,采用RT-PCR、Western blot以及免疫组化的方法检测组织中ANLN mRNA和蛋白表达水平,比较ANLN在肝内胆管癌组织与癌旁组织中的表达差异,分析其与肿瘤分期及肿瘤大小的关系。使用小干扰RNA建立ANLN低表达HUCCT1细胞系,行CCK8、平板克隆以及流式细胞检测观察ANLN对胆管癌细胞体外增殖能力的影响。行String软件预测与ANLN相关的蛋白,行RT-PCR、Western blot方法检测两者间的相关性以及周期蛋白CyclinD1表达差异。结果:Western blot和RT-PCR显示ANLN在肝内胆管癌组织中高表达。临床数据分析显示ANLN的表达与肝内胆管癌患者的肿瘤大小及TNM分期密切相关,并且ANLN表达高的患者预后较差。利用小干扰RNA(siRNA)下调ANLN能有效抑制胆管癌细胞的增殖能力。Western blot实验证明下调ANLN导致周期蛋白Cyclin D1、Cyclin A表达明显减少。String预测ANLN与TPX2相关,经Western blot和RT-PCR得到验证。结论:ANLN可能通过TPX2调节肝内胆管癌细胞的增殖能力,促进肝内胆管癌的发展,这可为肝内胆管癌的治疗提供新靶点。

Objective:To investigate the expression of ANLN in intrahepatic cholangiocarcinoma(ICC)and explored the function of ANLN in proliferation of ICC in vivo. Methods:We used the Western blot and real-time PCR(RT-PCR)to quantify the expression of ANLN in cancerous tissues and adjacent tissues of 40 patients with ICC. The differences in the expression of ANLN in ICC and the adjacent tissues of cancer were compared,and its relationship with tumor staging and tumor size was analyzed. ANLN low expression HUCCT1 cell lines were established using small interference RNA(siRNA). The cell proliferation was investigated by cell counting kit-8 assay(CCK-8),colony formation and cell cycle arrest. In addition,the proteins that regulate ANLN expression in CCA were selected by using String,and the relationship between them was verified by Western blot and RT-PCR. Results:The results of Western blot and RT-PCR revealed that ANLN was highly expressed in ICC tissues. Clinical data analysis showed that the expression of ANLN was closely related to the tumor size and TNM stage in patients with cholangiocarcinoma,and the prognosis of patients with high ANLN expression was poor. The reduction of ANLN by siRNA could effectively inhibit the proliferation ability of cholangiocarcinoma cells.Western blot showed that the reduction of ANLN led to a significant decrease in the expressions of periodic protein cyclin D1 and cyclin A. String showed that TPX2 may play a major role in the regulation of ANLN expression,Western blot and RT-PCR further validated the positive relationship between ANLN and TPX2. Conclusion:ANLN regulates the proliferation of cholangiocarcinoma cells,which promotes the development of ICC and provides a new target for the treatment of ICC.