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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 and mechanism of chemokine CX3C receptor 1 (CX3CR1) on human hepatocellular carcinoma Huh7 cells. Methods: The recombinant adenovirus Ad-si CX3CR1 targeting silencing of CX3CR1 was transfected into human hepatocellular carcinoma Huh7 cells. The expression of CX3CR1 in Huh7 cells was detected by RT-PCR and Western blotting. MTT, FCM, The effects of CX3CR1 expression silencing on the proliferation, apoptosis, migration and invasion of Huh7 cells were analyzed by the healing assay and Transwell chamber method respectively. The phosphoinositide 3-kinase (PI3K) -Akt signaling pathway was detected by Western blotting Related protein Akt and its phosphorylation levels. Meanwhile, Huh7 cells infected with Ad-si CX3CR1 were treated with different concentrations of PI3K inhibitor LY294002. Western blotting and Transwell invasion assay were used to detect the effect of LY294002 on CX3CR1-mediated Akt expression and cell invasion. Results: The CX3CR1 expression in Huh7 cells was significantly inhibited after Ad-si CX3CR1 infection (P <0.001), and the proliferation, migration and invasion ability of Huh7 cells were significantly enhanced (all P <0.001), while the apoptotic capacity was significantly decreased (P <0.01). The phosphorylation of Akt was increased in Huh7 cells after silence of CX3CR1 expression (P <0.001), and PI3K inhibitor LY294002 could effectively reverse the CX3CR1-mediated Akt phosphorylation and cell invasion (all P <0.001). Conclusion: The expression of CX3CR1 may promote the proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma Huh7 cells. The mechanism may be related to the activation of PI3K-Akt signaling pathway.