TLR4基因多态性对肝癌细胞HepG2增殖、迁移及凋亡的影响

摘要

目的:研究TLR4基因多态性对人肝癌细胞HepG2细胞增殖、迁移及凋亡的影响,并探讨其相关分子机制.方法:构建TLR4野生型(WT)、1196(C/T)位点及896(A/G)位点突变型质粒并将其稳转HepG2细胞株中;CCK-8法检测、Annexin V-PE/7-AAD流式细胞仪及Transwell小室实验检测TLR4基因多态性对HepG2细胞增殖、迁移及凋亡的影响;Western blot检测NF-κB p65(nuclear factorκB p65)表达水平的差异.结果:Western blot结果表明三组TLR4过表达细胞株中TLR4的含量显著高于正常组.与正常HepG2细胞相比,三组TLR4过表达细胞株增殖能力、迁移能力及p65表达量均增加;与TLR4野生型组相比,两组突变型细胞株增殖能力、迁移能力及p65表达量均减弱,且凋亡率增加,差异具有统计学意义(P<0.05).结论:TLR4可能通过影响NF-κB p65相关蛋白的表达促进肝癌细胞HepG2的增殖及迁移,其基因1196(C/T)位点及896(A/G)位点的突变会减弱肝癌细胞的增殖及迁移能力.%Objective:To study the effect of TLR4 gene polymorphism on the proliferation,migration and apoptosis of human hepatoma cell line HepG2 and to explore its related molecular mechanism.Methods:TLR4 wild type (WT),1196 (C/ T)and 896 (A/ G)mutant plasmids were constructed and transfected into HepG2 cell line.CCK-8 assay,Annexin V-PE/ 7-AAD flow cytometry and Transwell chamber were used to detect the effect of TLR4 over-expression and TLR4 gene polymorphism on the proliferation,migration and apoptosis of HepG2 cells.The expression of NF-κB p65 was detected by Western blot.Results:The results of Western blot showed that the content of TLR4 in TLR4 overexpressing cell lines was significantly higher than that in normal group.Compared with normal HepG2 cells,the proliferation ability,migration ability and p65 expression were increased in the 3 groups of TLR4 overex-pressing cell lines.Moreover,compared with the TLR4 wild type group,the proliferation ability,migration ability and p65 expression were decreased both in the two groups of mutant cell lines,the difference was significant (P< 0.05).Conclusion:TLR4 might promote the proliferation and migration of hepatoma cell line HepG2 by affecting the expres-sion of NF-κB p65-related protein.Mutations in TLR4 gene 1196 (C/ T)and 896 (A/ G)sites attenuated the pro-liferation and migration of hepatoma cells.