趋化因子受体CX3CR1表达沉默对人肝细胞癌Huh7细胞的作用及机制研究  

Effects of expression silencing of chemokine receptor CX3CR1 on human hepatocellular carcinoma Huh7 cells and its machanism

作　　者：何方[1] 张汝益[1] 王静[1] 邓芳[1] 范梦恬 李亚[1] 郭杨柳 施琼[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《肿瘤》2015年第1期46-54,共9页Tumor

摘　　要：目的:探讨趋化因子CX3C受体1(chemokine CX3C receptor 1,CX3CR1)对人肝细胞癌Huh7细胞的作用及其机制。方法:将靶向沉默CX3CR1表达的重组腺病毒Ad-si CX3CR1感染至人肝细胞癌Huh7细胞,然后应用RT-PCR和蛋白质印迹法检测Huh7细胞中CX3CR1的表达水平,采用MTT、FCM、划痕愈合实验和Transwell小室法分别分析CX3CR1表达沉默对Huh7细胞增殖、凋亡、迁移和侵袭的影响,再通过蛋白质印迹法检测磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)-Akt信号通路相关蛋白Akt及其磷酸化水平的变化。同时,用不同浓度的PI3K抑制剂LY294002作用感染了Ad-si CX3CR1的Huh7细胞后,分别采用蛋白质印迹法和Transwell侵袭实验检测LY294002对CX3CR1介导的Akt表达和细胞侵袭能力的影响。结果:感染Ad-si CX3CR1后,肝细胞癌Huh7细胞中CX3CR1表达被明显抑制(P<0.001),细胞的增殖、迁移和侵袭能力均明显增强(均P<0.001),凋亡能力则明显降低(P<0.01)。沉默CX3CR1表达后,Huh7细胞中Akt的磷酸化水平升高(P<0.001),且PI3K抑制剂LY294002能有效逆转CX3CR1介导的Akt磷酸化和细胞侵袭(均P<0.001)。结论:沉默趋化因子受体CX3CR1表达可以促进人肝细胞癌Huh7细胞的增殖、迁移和侵袭,并抑制细胞凋亡,其机制可能与活化PI3K-Akt信号通路有关。Objective：To investigate the effect of chemokine CX3 C receptor 1（CX3CR1） on human hepatocellular carcinoma Huh7 cells and its probable machanism.Methods：Hepatocellular carcinoma Huh7 cells were transfected with the recombinant adenovirus Ad-siCX3 CRl targeting and silencing CX3CR1 gene.Then the expression of CX3CR1 was detected by reverse transcription-PCR（RT-PCR） and Western blotting.The proliferation,apoptosis,migration and invasion of Huh7 cells were tested by MTT,flow cytometry,wound-healing and Transwell assay,respectively.The expression and phosphorylation of Akt which was related to phosphoinositide 3-kinase（PI3K）-Akt signal pathway were detected by Western blotting.Meanwhile,Huh7 cells transfected with AdsiCX3 CRl were treated with different concentrations of PI3 K inhibitor LY294002,then the expression of Akt and cell invasion ability induced by CX3CR1 were detected by Western blotting and Transwell assay,respectively.Results：After Ad-siCX3 CRl was stably transfected into Huh7 cells,the expression of CX3CR1 was significantly inhibited（P〈0.001）,and the cell proliferation and migration and invasion abilities were significantly increased（P〈0.001）,but the apoptosis rate was significantly decreased（P0.01）.When the expression of CX3CR1 was silenced,the level of phosphorylated Akt（p-Akt） in Huh7 cells was increased（P〈0.001）,and LY294002 could reverse CX3CR1-induced phosphorylation of Akt and invasion of Huh7 cells effectively（P〈0.001）.Conclusion：CX3CR1 gene silencing can promote proliferation,migration and invasion of hepatocellular carcinoma Huh7 cells,and also can suppress their apoptosis.The activation of PI3K-Akt signaling pathway may be involved in this process.

关 键 词：癌 肝细胞 基因沉默 受体 趋化因子CX3CL1 细胞增殖 细胞凋亡 细胞迁移分析 癌基因蛋白质v-akt 

分 类 号：R735.7[医药卫生—肿瘤]