摘要
目的探讨Hedgehog信号通路关键蛋白Gli1对人肝癌细胞中Twist1蛋白表达和细胞迁移的影响。方法应用蛋白免疫印迹技术在Huh7、Hep G2、PLC、SMMC-7721、Bel-7404五种人肝癌细胞中筛选Gli1高表达细胞系,根据Gen Bank数据库设计并合成3条Gli1-siRNA经质粒转染到已筛选出Gli1高表达的人肝癌细胞中,分别为p GCsi-U6-GLi1siRNA-1(siRNA-1组)、p GCsi-U6-GLi1siRNA-2(siRNA-2组)、p GCsi-U6-GLi1siRNA-3(siRNA-3组),而空白对照组(N组)不转染任何序列,阴性对照组转染阴性对照序列(NC组)。转染48 h后分别使用实时荧光定量PCR和Western blotting法检测各组Gli1mRNA和蛋白表达,筛选出最佳siRNA序列。对筛选出的最佳siRNA序列组的细胞,采用细胞划痕实验观察其迁移能力,用Western blotting检测该组细胞中Twist1、E-Cadherin蛋白表达。结果Western blotting检测结果提示,与另外4种人肝癌细胞比较,Bel-7404细胞株中Gli1表达量增加(P均<0.05);与其他组比较,siRNA-2组中的Gli1表达量最低(P均<0.05);与N组比较,siRNA-2组中的Twist1蛋白表达下调,E-cadherin表达上调,N-Cadherin和Vimentin表达下调,迁移能力下降(P均<0.05)。结论 Gli1可上调肝癌Bel-7404细胞中Twist1蛋白表达,促进人上皮-间质细胞转化及迁移。
Objective To explore the effects of the Hedgehog signal pathway crucial protein Gli1 Hedgehog signal pathway on the expression of Twist1 protein and migration of human hepatoma cells.Methods The cell lines with highly expressed Gli1 were screened out from the human hepatoma cell lines Huh7,hepG2,PLC,SMMC-7221,and Bel-7404 by Western blotting.According to the biological information of GenBank database,three Gli-siRNA were transfected into the screened human hepatoma cells with highly expressed Gli1 via plasmids:pGCsi-U6-GLi1siRNA-1(siRNA-1 group),pGCsi-U6-GLi1siRNA-2(siRNA-2 group),and pGCsi-U6-GLi1siRNA-3(siRNA-3 group).The cells in the blank control group(group N)were not transfected with any siRNA sequence,and the cells in the negative control group(NC group)were transfected with the negative control sequence.The mRNA and protein expression of Gli after 48-hour transfection was assessed by qRT-PCR and Western blotting to screen out the most optimal siRNA sequence.The migration ability and viability of the cell lines with the most optimal siRNA sequence were detected by Scratch test.The protein expression of Twist1 and E-cadherin of cells was measured by Western blotting.Results Western blotting showed that compared with the other four human hepatoma cell lines,the protein expression of Gli1 in the Bel-7404 cells was significantly up-regulated(all P<0.05);compared with the other groups,the expression of Gli1 in the siRNA-2 group was significantly the lowest(all P<0.05);compared with the group N,the protein expression of Twist11 was down-regulated while the expression of E-cadherin was up-regulated,the expression of N-Cadherin and Vimentin was down-regulated,and the migration ability significantly decreased in the siRNA-2 group(all P<0.05).Conclusion Silencing the expression of the Gli1 with the siRNA can up-regulate the expression of Twist1 in the Bel-7404 cells,and promote the transformation and migration of epithelial-mesenchymal cells.
作者
甘鲁慧
吕沐瀚
邓明明
GAN Luhui;LYU Muhan;DENG Mingming(Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)
出处
《山东医药》
CAS
2018年第9期1-4,共4页
Shandong Medical Journal
基金
国家自然科学基金面上项目(81672458)
四川省教育厅科研基金一般项目(16ZB0197)
