幽门螺杆菌感染通过核因子κB信号通路调控Fas相关因子1表达的相关机制

Mechanism for Helicobacter pylori infection to regulate Fas-associated factor 1 expression through nuclear factor-κB signaling pathway

目的:探讨幽门螺杆菌(Helicobacter pylori,H.pylori)感染对敲除核因子-κB(nuclear factor-κB,NF-κB)通路上IKKβ、p65基因后过表达Fas相关因子1(Fas-associated factor 1,FAF1)胃癌细胞的影响,进一步明确H.pylori致癌的相关机制.方法:构建针对IKKβ、p65的siRNA慢病毒载体(LV-IKKβ-RNAi、LV-p65-RNAi),转染过表达FAF1的HGC-27细胞,实时定量聚合酶链反应(RT-PCR)和蛋白质印迹(Western blot)法检测转染前后IKKβ、p65、FAF1 mRNA和蛋白表达.应用CCK8法检测转染后细胞增殖能力的变化.用H.pylori培养滤液感染基因敲除细胞,用RT-PCR和Western blot法检测H.pylori感染前后IKKβ、p65、FAF1 mRNA和蛋白表达.结果:成功将LV-IKKβ-RNAi、LV-p65-RNAi和阴性对照(LV-NC-RNAi)转染入过表达FAF1胃癌细胞,转染后72 h,LV-IKKβ-RNAi和LV-p65-RNAi组IKKβ和p65 mRNA和蛋白表达显著低于LV-NC-RNAi组和未转染组(均P<0.01);转染前后各组间FAF1 mRNA和蛋白表达无显著差异(P>0.05).LV-IKKβ-RNAi组和LV-p65-RNAi组细胞增殖能力升高(P<0.01).H.pylori感染后LV-IKKβ-RNAi组和LV-p65-RNAi组IKKβ、p65 mRNA和蛋白表达与感染前相比无显著差异(P>0.05),而LV-NC-RNAi组和未转染组IKKβ、p65 mRNA和蛋白表达都显著高于感染前(P<0.01).H.pylori感染后LV-IKKβ-RNAi组和LV-p65-RNAi组FAF1 mRNA和蛋白表达与感染前相比无显著差异(P>0.05),而LV-NC-RNAi组和未转染组FAF1 mRNA和蛋白表达显著低于感染前(P<0.01).结论:H.pylori感染可能通过介导NF-κB信号通路下调抑癌因子FAF1的表达,进而导致胃癌的发生.

AIM： To explore the influence of Helicobacter pylori（H. pylori） infection on human gastric cancer cells overexpressing Fas-associated factor 1（FAF1） after knockout of IKKβ or p65 of the nuclear factor-κB（NF-κB） pathway, in order to further clarify the mechanism of H. pylori in gastric carcinogenesis.METHODS： Lentivirus vectors carrying siRNA targeting IKKβ or p65 were constructed（LV-IKKβ-RNAi, LV-p65-RNAi） and used to transfecting human gastric cancer cells HGC-27 overexpressing FAF1. IKKβ, p65, and FAF1 mRNA and protein expression was detected by real-time PCR and Western blot before and after transfection. CCK8 assay was applied to detect cell proliferation after transfection. The transfected cells were infected with H. pylori culture filtrate, and real-time PCR and Western blot were applied to detect IKKβ, p65 and FAF1 expression before and after H. pylori infection.RESULTS： LV-IKKβ-RNAi, LV-p65-RNAi and negative control（LV-NC-RNAi） were transfected into HGC-27 cells overexpressing FAF1 successfully. After transfection for 72 h, the expression of IKKβ and p65 mRNA and proteinin the LV-IKKβ-RNAi group and LV-p65-RNAi group were significantly lower than that in the LV-NC-RNAi group and untransfected group（P 0.01）. There was no statistically significant difference in the expression of FAF1 mRNA and protein in the four groups（P 0.05）. The proliferation of cells in the LV-IKKβ-RNAi group and LV-p65-RNAi group increased. H. pylori culture filtrate was used to infect different groups of cells. There was no statistically significant difference in the expression of IKKβ and p65 mRNA and protein in the LV-IKKβ-RNAi group and LV-p65-RNAi group before and after H. pylori infection（P 0.05）, but the expression of IKKβ and p65 mRNA and protein in the LV-NC-RNAi group and untransfected group after H. pylori infection was significantly higher than that before H. pylori infection（P 0.01）. There was no statistically significant difference in the expression of FAF1 mRNA and protein in the LV-IKKβ-RNAi group and LV-p65-RNAi group before and after H. pylori infection（P 0.05）, but the expression of FAF1 mRNA and protein in the LV-NC-RNAi group and untransfected group after H. pylori infection was significantly lower than that before H. pylori infection（P 0.01）.CONCLUSION： H. pylori infection might regulate FAF1 expression through the NF-κB signaling pathway, and downregulation of FAF1 could lead to gastric carcinogenesis.