siRNA靶向沉默ZFX基因对肝内胆管癌细胞生物学功能的影响

Effect of targeted siRNA silencing ZFX gene on biological function of intrahepatic cholangiocarcinoma cells

目的探讨siRNA沉默X染色体连锁的锌指蛋白(ZFX)基因的表达后对肝内胆管癌细胞(HCCC-9810)的增殖、克隆、迁移及侵袭能力的影响。方法通过脂质体lipofectamine 3000介导转染肝内胆管癌细胞株HCCC-9810,抑制ZFX基因在肝内胆管癌细胞系中的表达,RT-PCR检测ZFX基因的mRNA表达水平的变化,Western blot检测ZFX基因的蛋白表达水平的变化。ZFX基因下调后分别用软琼脂克隆形成实验检测克隆形成能力,CCK-8增殖实验检测细胞增殖情况,Transwell检测细胞的迁移、转移情况。结果RT-PCR及Western blot结果表明与Cell组、NC-siRNA组相比,ZFX-siRNA组有效降低了HCCC-9810细胞系ZFX的mRNA水平及蛋白水平的表达。克隆形成实验显示:ZFX基因沉默后,该细胞系克隆形成能力明显减弱。CCK-8增殖实验证明实验组细胞增殖能力明显降低。Transwell实验显示,Cell组、NC-siRNA组细胞迁移、转移能力明显高于ZFX-siRNA组细胞。各组实验中Cell组、NC-siRNA组之间均未见明显差异。结论本实验siRNA成功降低肝内胆管癌细胞系HCCC-9810中ZFX基因的表达并证明ZFX的表达降低后与肝内胆管癌细胞的克隆形成、增殖、迁移、转移能力相关。推测ZFX在肝内胆管癌的发生发展中发挥重要的作用,可作为该基因治疗的新靶点。

Objective To explore the effect of siRNA silencing ZFX gene on proliferation,migration and cycle of intrahepatic cholangiocarcinoma cells(HCCC-9810).Methods Lipofectamine 3000 was used to transfect the intrahepatic cholangiocarcinoma cell line HCCC-9810 to inhibit the expression of ZFX gene in intrahepatic cholangiocarcinoma cell lines.The mRNA expression level of ZFX gene was detected by RT-PCR,and the protein expression level of ZFX gene was detected by Western blot.After the down-regulation of ZFX gene,the ability of colony formation was detected by the soft lipid agarose formation assay.CCK-8 proliferation assay was used to detect cell proliferation,and Transwell was used to detect cell migration and metastasis.Results RT-PCR results showed that ZFX-siRNA effectively reduced the expression of ZFX mRNA in HCCC-9810 cell line compared to Cell,NC-siRNA group.Clone formation experiments showed that the clonogenic capacity of this cell line was significantly weakened after ZFX silencing.CCK-8 proliferation assay showed that the experimental group cell proliferation decreased significantly.Transwell experiments showed that Cell,NC-siRNA group cell migration and metastasis was significantly higher than ZFX-siRNA cells.Conclusion The siRNA of this experiment successfully reduced the expression of ZFX gene in intrahepatic cholangiocarcinoma cell line HCCC-9810 and demonstrated that the decreased expression of ZFX is related to the ability of intrahepatic cholangiocarcinoma cell clone formation,proliferation,migration and metastasis.It is speculated that ZFX plays an important role in the occurrence and development of intrahepatic cholangiocarcinoma and can be used as a new target for this gene therapy.