PLK1稳定β-catenin 促进食管鳞癌细胞上皮间质转化

PLK1 promotes epithelial-mesenchymal transition of esophageal squamous cell carcinoma cells by stabilizing β-catenin

目的研究PLK1(Polo-like kinase 1)对食管鳞癌细胞株TE-15上皮间质转化(epithelial-mesenchymal transition,EMT)的影响,并探讨其作用机制。方法 Western blot检测PLK1稳定过表达的食管鳞癌细胞克隆与空载对照克隆中EMT相关标志蛋白E-钙粘素(E-cadherin)、波形蛋白(vimentin)的表达情况;Real-time PCR检测vimentin mRNA表达水平;分别提取细胞总蛋白和胞核蛋白,Western blot检测PLK1对信号通路分子β-连环蛋白(β-catenin)表达的影响;采用RNA干扰技术,在PLK1过表达克隆中分别瞬时转染靶向β-catenin的siRNA和非特异性siRNA,Western blot检测细胞中vimentin的表达;通过免疫沉淀和Western blot检测PLK1对β-catenin降解复合物的影响。结果与空载对照克隆相比,PLK1过表达的食管鳞癌细胞中,间质标志物vimentin的表达明显上调,上皮标志物E-cadherin的表达明显下调,提示食管鳞癌细胞发生了EMT;vimentin mRNA表达水平亦明显升高;在PLK1过表达的克隆中,细胞总蛋白和胞核蛋白中β-catenin表达都明显增加,敲降β-catenin的表达能引起vimentin的表达下降;Axin免疫沉淀物中的APC及GSK-3β都明显减少。结论 PLK1可能通过抑制β-catenin降解复合物的聚合而稳定β-catenin,从而上调vimentin表达,促进食管鳞癌细胞TE-15发生EMT。

Aim To investigate the effect of PLK1 on epithelial-mesenchymal transition （EMT）of human e-sophageal squamous cell carcinoma （ESCC）cells TE-1 5 and its relevant molecular mechanisms.Methods PLK1 overexpressed ESCC cells and control vector were used as the experimental cells.The expression of EMT-related protein markers E-cadherin and vimentin were measured by Western blot.vimentin mRNA was measured by Real-time PCR.Total cellular protein and nuclear protein were respectively extracted,and then they were used to detect the expression of β-catenin by Western blot.β-catenin siRNA and non-specific siR-NA were transiently transfected into the cell clones overexpressed PLK1 ,and then vimentin was detected by Western blot.β-catenin protein degradation com-plex was detected by immunoprecipitation and Western blot.Results The mesenchymal marker vimentin was＆amp;nbsp;distinctively upregulated and the epithelial marker E-cadherin was distinctively downregulated in the cell clones overexpressed PLK1 ,compared with those in the vector clones.This indicated that EMT occurred in ESCC cells.vimentin mRNA was also markedly in-creased.In the cell clones overexpressed PLK1 ,β-catenin were both elevated from the total cells and the nucleus.The expression of vimentin was reduced whenβ-catenin was knocked down.APC and GSK-3βwere both reduced from Axin immunoprecipitate in the cell clones overexpressed PLK1 .Conclusion PLK1 up-regulates vimentin and promotes EMT in ESCC cells probably by inhibiting the formation of protein degrada-tion complex and stabilizing β-catenin.