核磷素1通过激活上皮-间质转化信号通路促进胆管癌细胞增殖、迁移和侵袭能力

NPM1 promotes cell proliferation,migration and invasion of cholangiocarcinomacell lines by activating the epithelial-mesenchymal transition pathway

目的探索核磷素1(NPM1)基因对胆管癌细胞系Huh28和RBE的细胞增殖、迁移和侵袭能力的影响,并探讨NPM1影响胆管癌的作用机制。方法采用瞬时转染将包含NPM1基因的过表达重组质粒转染胆管癌细胞系Huh28和RBE以过表达NPM1,采用小干扰RNA(siRNA)瞬时转染Huh28和RBE以敲低NPM1,并使用Western印迹实验检测其中NPM1蛋白的表达水平,以验证过表达和敲低NPM1的效果。采用CCK-8法和Transwell法分别检测细胞增殖和迁移能力;采用实时定量PCR(qPCR)及Western印迹实验,检测上皮-间质转化(EMT)过程中相关分子的mRNA及蛋白表达水平,包括钙黏蛋白1(CDH1)、钙黏蛋白2(CDH2)、β连环蛋白1(CTNNB1)和波形蛋白(VIM);采用Log-rank检验比较NPM1高、低表达组胆管癌患者的生存期差异。结果敲低NPM1可抑制Huh28和RBE细胞的增殖、迁移及侵袭能力,而过表达NPM1则促进Huh28和RBE细胞的增殖、迁移及侵袭能力。过表达NPM1促进Huh28和RBE细胞的EMT过程,而敲低NPM1则抑制Huh28和RBE细胞的EMT过程。NPM1在胆管癌组织中上调表达,NPM1的表达水平与胆管癌患者的生存期无相关性。结论NPM1通过激活EMT信号通路促进胆管癌细胞系的增殖、迁移和侵袭能力,在胆管癌的发生发展中可能发挥癌基因的功能。

Objective To investigate the effects of nucleophosmin 1(NPM1)on the proliferation and migration of cholangiocarcinoma cell lines Huh28 and RBE,and to explore the potential mechanism.Methods Recombined plasmid containing NPM1 was transiently transfected into Huh28 and RBE cells for overexpression of NPM1.Small interfering RNA(siRNA)was used to knock down the endogenous NPM1 in Huh28 and RBE cells.To verify the effects of overexpression and knockdown of NPM1,NPM1 protein expression levels were detected in Huh28 and RBE by Western blotting assays.CCK-8 assays were used to assess the proliferation of Huh28 and RBE cells.The effects of NPM1 on migration and invasion of Huh28 and RBE cells were verified by Transwell assays.Quantitative real time polymerase chain reaction(qPCR)and Western blotting assays were performed to detect the mRNA and protein expression levels of specific genes in epithelial-mesenchymal transition(EMT),including cadherin 1(CDH1),cadherin 2(CDH2),β-catenin1(CTNNB1)and vimentin(VIM).Log-rank test was used to reveal the difference in survival between cholangiocarcinoma patients with high and low NPM1 expressions.Results Knockdown of NPM1 reduced the proliferation,migration and invasion of Huh28 and RBE cells,whereas overexpression of NPM1 induced the proliferation,migration and invasion of Huh28 and RBE cells.Overexpression of NPM1 induced EMT,whereas knockdown of NPM1 reduced EMT.NPM1 was up-regulated in cholangiocarcinoma tissues compared with non-tumor liver tissues,and its high expression was marginally associated with poor prognosis in patients with cholangiocarcinoma.Conclusion NPM1 can promote the proliferation,migrationand invasion of cholangiocarcinoma cell lines by activating the EMT pathway.Thus,NPM1 might act as an oncogene in the development of cholangiocarcinoma.