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    阿瑞匹坦抑制SFN蛋白对非小细胞肺癌细胞上皮间质转化机制研究

    Effect of aprepitant on epithelial-mesenchymal transformation in non-small cell lung cancer through inhibiting SFN protein

    • 摘要: 目的 研究分层蛋白Stratifin(SFN)对非小细胞肺癌A549细胞上皮间质转化(EMT)和迁移、侵袭的调控作用,以及阿瑞匹坦对SFN功能的影响。方法 通过瞬时转染技术将siSFN-1、siSFN-2、SFN、NC基因转入A549细胞中,Western blot实验检测SFN、E-钙黏蛋白、N-钙黏蛋白以及波形蛋白表达水平,细胞划痕实验和Transwell实验检测细胞迁移、侵袭作用;通过CCK8实验检测阿瑞匹坦对过表达SFN的A549细胞在24、48、72 h增殖的调控能力;根据CCK8测得的药物IC50值进一步探索阿瑞匹坦不同浓度(16、8、4μmol/L)作用于过表达SFN的A549细胞48 h后的SFN、E-钙黏蛋白、N-钙黏蛋白、波形蛋白的表达以及细胞迁移、侵袭的变化。结果 24、48、72 h的细胞增殖-毒性实验中,随着阿瑞匹坦作用于细胞的时间不断延长,细胞增殖能力也随之出现逐渐下降趋势,药物抑制作用显著(P<0.05)。划痕实验结果表明转染SFN基因的A549细胞迁移距离明显高于NC、siSFN-1、siSFN-2组(P<0.05)。安慰剂组的迁移距离较16、8、4μmol/L阿瑞匹坦组迁移距离长;并且在16、8、4μmol/L瑞匹坦组中,细胞迁移距离随浓度降低逐渐加长(P<0.05)。Transwell迁移和侵袭实验中转染SFN的A549细胞迁移和侵袭的细胞数量多于NC、siSFN-1、siSFN-2组(P<0.05)。安慰剂处理组的细胞迁移和侵袭数量较16、8、4μmol/L阿瑞匹坦组多;并且在16、8、4μmol/L瑞匹坦组中,细胞迁移和侵袭的数量随着阿瑞匹坦浓度降低逐渐增多(P<0.05)。Western blot分析显示过表达SFN促使上皮标志物E-钙黏蛋白减少,间质标志物N-钙黏蛋白和波形蛋白增加(P<0.05)。安慰剂组E-钙黏蛋白较不同浓度阿瑞匹坦组减少,N-钙黏蛋白和波形蛋白较不同浓度阿瑞匹坦组明显增多;阿瑞匹坦组中随浓度增高E-钙黏蛋白增多,N-钙黏蛋白减少(P<0.05)。结论 过表达SFN能够促进非小细胞肺癌的上皮间质转化和迁移、侵袭,而阿瑞匹坦可以通过抑制SFN表达进而抑制其介导的上皮间质转化和迁移、侵袭。

       

      Abstract: Objective To investigate the regulatory effects of stratifin(SFN) on the epithelial-mesenchymal transformation, migration, and invasion of non-small cell lung cancer A549 cells, and the effects of aprepitant on SFN function.Methods First, siSFN-1, siSFN-2, SFN and NC genes were transferred into A549 cells by transient transfection. The expression of SFN, E-cadherin, N-cadherin and vimentin were detected by Western blot. The cell migration and invasion were detected by the wound healing assay and Transwell assay. The regulatory effect of aprepitant on the proliferation of SFN-overexpressed A549 cells was measured by CCK8 assay at 24, 48 h and 72 h.According to the IC50 value measured by CCK8 assay, the expression of SFN, E-cadherin, N-cadherin, vimentin in A549 cells overexpressing SFN for 48 h, as well as cell migration and invasion changes were further explored after treatment with aprepitant at the concentrations of 16, 8μmol/L and 4 μmol/L.Results In the cell proliferation-toxicity test at 24, 48 h and 72 h, with the extension of aprepitant treatment time, the cell proliferation ability showed a gradual decline, with significant drug inhibitory effect(P<0.05).Wound healing assay results showed that the migration distance of SFN transfected A549 cells was significantly longer than that of the NC, siSFN-1 and siSFN-2 groups(P<0.05).The migration distance of the placebo group was longer than that of the 16, 8 μmol/L and 4 μmol/L aprepitant group, and the migration distance of the cells in the 16, 8 μmol/L and 4 μmol/L aprepitant groups gradually increased with the decrease of the concentrations(P<0.05).In Transwell migration and invasion experiments, the number of migrated and invaded A549 cells transfected with SFN was more than those in the NC, siSFN-1 and siSFN-2 groups(P<0.05).The number of migrated and invaded cells in the placebo group was more than those in the 16, 8 μmol/L and 4 μmol/L aprepitant groups, and the number of migrated and invaded cells in the 16, 8 μmol/L and 4 μmol/L aprepitant groups gradually increased with the decrease of concentrations(P<0.05). According to Western blot analysis, overexpression of SFN decreased the epithelial markers E-cadherin and increased the interstitial markers N-cadherin and vimentin(P<0.05).Theplacebo group showed decreases in E-cadherin, and increases in N-cadherin and vimentin, compared with the three other groups, and E-cadherin increased and N-cadherin decreased with the increase of concentrations intheAprepitant group(P<0.05).Conclusions SFN overexpression can promote epithelial mesenchymal transformation, migration and invasion in non-small cell lung cancer.Aprepitant can inhibit SFN expression and thereby inhibit its mediated epithelial mesenchymal transformation, migration and invasion.

       

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