Dickkopf-1沉默通过下调β-catenin水平抑制胃癌细胞侵袭及上皮-间充质转化

摘要

目的: 探讨分泌蛋白Dickkopf-1 (DKK1)在人胃癌细胞中的表达及其对胃癌细胞侵袭能力的影响.方法: 以real-time PCR和Western blot法检测DKK1在人胃黏膜细胞(GES-1)和胃癌细胞(MKN-45和SGC-7901)中的表达水平;以RNA干扰法沉默DKK1,沉默效果以real-time PCR、Western blot及ELISA法验证;Transwell实验检测细胞侵袭能力,以丝裂霉素C抑制细胞增殖;real-time PCR及Western blot法检测细胞E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)及β-连环蛋白(β-catenin)水平.结果: DKK1在MKN-45和SGC-7901细胞中的表达明显高于GES-1细胞,表明DKK1在胃癌细胞中表达显著升高;DKK1在MKN-45和SGC-7901细胞中被成功沉默后,细胞侵袭能力显著下降,并具有时间依赖性,同时伴随E-cadherin表达增高及N-cadhe-rin和vimentin表达下降,表明DKK1沉默能够显著抑制胃癌细胞侵袭和上皮-间充质转化(epithelial-mesenchymal transition,EMT);经外源性重组DKK1(rDKK1)转染肿瘤细胞后,进一步证实DKK1具有促胃癌细胞侵袭的作用,并可促进EMT进程;DKK1沉默通过下调β-catenin水平来实现其对胃癌细胞侵袭及EMT的抑制作用.结论: DKK1在人胃癌细胞中表达显著增高,且DKK1沉默能够通过下调β-catenin水平抑制胃癌细胞侵袭及EMT过程.%AIM: To explore the expression of Dickkopf-1 (DKK1) in human gastric carcinoma cells, and the influences of DKK1 gene silencing on cell invasion.METHODS: The levels of DKK1 in the human gastric mucosa cell line GES-1 and gastric carcinoma cell lines MKN-45 and SGC-7901 were detected by real-time PCR and Western blot.DKK1 gene was silenced by RNA interference, which was verified by real-time PCR, Western blot and ELISA.The cell invasion ability was determined by Transwell assay, and the cell proliferation was inhibited by mitomycin C.The levels of E-cadherin, N-cadherin, vimentin and β-catenin were determined by real-time PCR and Western blot.RESULTS: The expression of DKK1 was significantly higher in MKN-45 cells and SGC-7901 cells than that in GES-1 cells, indicating that DKK1 expression was obviously increased in gastric carcinoma cells.After successful silencing of DKK1 gene in the MKN-45 cells and SGC-7901 cells, the cell invasion ability was markedly decreased in a time-dependent pattern with increased expression of E-cadherin and decreased expression of N-cadherin and vimentin, indicating that DKK1 silencing dramatically inhibited gastric carcinoma cell invasion and epithelial-mesenchymal transition (EMT).The introduction of exogenous recombinant DKK1 (rDKK1) demonstrated the promoting effect of DKK1 on gastric carcinoma cell invasion and EMT.In addition, the inhibitory effects of DKK1 silencing on gastric carcinoma cell invasion and EMT were fulfilled by down-regulating β-catenin.CONCLUSION: The expression of DKK1 is significantly increased in human gastric carcinoma cells.Silencing of DKK1 markedly inhibits gastric carcinoma cell invasion and EMT by down-regulating β-catenin.