HVEM基因干扰后非小细胞肺癌细胞的迁移、侵袭能力变化及其机制

Effects of HVEM gene silencing on migration and invasion of non-small-cell lung cancer cells

目的 观察疱疹病毒侵入介体(HVEM)基因干扰对非小细胞肺癌(NSCLC)细胞迁移、侵袭能力的影响,并探讨其可能的作用机制。方法 通过Western blotting法检测正常肺上皮细胞BEAS-2B和NSCLC`细胞系SKMES-1、H1299、H460、H226、H520中HVEM蛋白表达,筛选出HVEM蛋白表达升高最明显的H1299细胞进行后续实验。将H1299细胞分为si-NC组、si-HVEM-1组和si-HVEM-2组,分别用Lipofectamine 2000瞬时转染si-NC、siHVEM-1和si-HVEM-2干扰序列,转染48 h。采用Transwell小室实验检测细胞迁移和侵袭能力,采用实时荧光定量PCR法和Western blotting法检测HVEM、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、β-连环蛋白(β-catenin)mRNA和蛋白相对表达量。结果 与si-NC组比较,si-HVEM-1组和si-HVEM-2组迁移、侵袭细胞数及HVEM mRNA、蛋白相对表达量均减少,且si-HVEM-2组减少更明显(P均<0.05)。与si-NC组比较,siHVEM-1和si-HVEM-2组E-cadherin mRNA及蛋白相对表达量均升高,N-cadherin、Vimentin、β-catenin mRNA及蛋白相对表达量均降低,si-HVEM-2组E-cadherin、Vimentin、β-catenin mRNA相对表达量及Vimentin、β-catenin蛋白相对表达量变化更明显(P均<0.05)。结论 干扰HVEM基因会降低NSCLC细胞的迁移和侵袭能力,其机制可能与通过抑制Wnt/β-catenin信号通路活化而抑制上皮—间质转化的发生有关。

Objective To investigate the effects of HVEM gene silencing on migration and invasion of non-smallcell lung cancer(NSCLC)cells,and to explore the mechanism.Methods The protein expression of HVEM in normal lung epithelial cell line BEAS-2B and NSCLC cell lines SK-MES-1,H1299,H460,H226 and H520 was detected by Western blotting.H1299 cells with the highest protein expression of HVEM were selected as the subsequent experiments.H1299 cells were cultured in vitro and randomly divided into the si-NC group(transfected with Lipofectamine 2000 and NC sequence),si-HVEM-1 group(transfected with Lipofectamine 2000 and si-HVEM-1 sequence),and si-HVEM-2group(transfected with Lipofectamine 2000 and si-HVEM-2 sequence).Transwell assay was used to detect cell migration and invasion at 48 h of transfection.The relative mRNA and protein expression levels of HVEM,E-cadherin,N-cadherin,Vimentin and β-catenin were detected by q RT-PCR and Western blotting,respectively.Results Compared with the siNC group,the number of migrating and invading cells and the relative mRNA and protein expression levels of HVEM decreased in the si-HVEM-1 and si-HVEM-2 groups,and the decrease was more obvious in the si-HVEM-2 group(all P<0.05).Compared with the si-NC group,the relative m RNA and protein expression levels of E-cadherin in the si-HVEM-1and si-HVEM-2 groups increased(all P<0.05),while the relative mRNA and protein expression levels of N-cadherin,Vimentin,and β-catenin decreased,the changes of E-cadherin,Vimentin and β-catenin mRNA expression and Vimentin and β-catenin protein expression in the si-HVEM-2 group were more obvious(all P<0.05).Conclusion HVEM gene silencing can inhibit the migration and invasion of NSCLC cells,which may be related to the suppression of epithelial-mesenchymal transition by inhibiting of activation of Wnt/β-catenin signaling pathway.