The Research Of Regulatory Role Of Gene Silencing Of Ikkbeta And P65 On NF-Kappab Pathways For Gastric Cancer Cells Overexpressing FAF1

OBJECTIVETo explore the influence of gene silencing of IKKβ or p65 in nuclear factor-κB (NF-κB) signaling pathways on gastric cancer cells HGC-27 overexpressing Fas associated factor 1 (FAF1), and the role of NF-κB signaling pathways in Helicobacter pylori (Hp) infection related gastric cancer. And to further clarify the correlation between FAF1 and NF-κB signaling pathways in Hp related gastric cancer.METHODSsiRNA lentivirus vector targeted IKKβ or p65 was constructed respectively (LV-IKKβ-RNAi, LV-p65-RNAi) and transfected into human gastric cancer cells HGC-27 overexpressing FAF1. IKKβ, p65, FAF1 mRNA and protein expression were detected by Quantitative Real-time PCR (qRT-PCR) and Western blot (WB) before and after transfection. CCK8 assay was applied to detect cell proliferation after transfection. Finally, the transfected cells were infected with Hp culture filtrate, and Real time PCR and Western blot were applied to detect IKKβ,p65 and FAF1 expression before and after Hp infection. And then Western blotting (WB) was performed to detect the protein level of IKKα/β, p65, IκBα and the corresponding phosphorylation genes (P-IKKα/β, P-p65, P-IκBα) on NF-κB signaling pathways after Hp infection, and ELISA was applied to detect the concentration of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8).RESULTS1. siRNA lentivirus vector targeted IKKβ or p65 (LV-IKKβ-RNAi, LV-p65-RNAi) was constructed successfully. The results of sequence determination showed that the constructed recombinant sequence were correct.2. The concentrate of lentivirus was packaged successfully, and after lentivirus transfected 293T cell, a lot of red flurorescence was observed. The results of flurorescence method measuring lentivirus titer showed rhat the titer of LV-IKKβ-RNAi was 3×108 TU/ml, the titer of LV-p65-RNAi was 6×108 TU/ml, and the titer of LV-NC-RNAi was 5×108 TU/ml.3. After transfected with IKKβ or p65 siRNA respectively, the spindle cells became irregular in LV-IKKβ-RNAi group and LV-p65-RNAi group, and cells in LV-NC-RNAi group did not change significantly. After transfection for 72 hours, the transfection efficiency observed by fluorescence microscope was up to 90%.4. After transfected with IKKβ or p65 siRNA respectively, the expression of IKKβ and p65 mRNA in LV-IKKβ-RNAi group and LV-p65-RNAi group were significantly lower than LV-NC-RNAi group and untransfected group (all P<0.01). There was no statistically significant difference about the expression of IKKβ and p65 mRNA in LV-NC-RNAi group and untransfected group (P> 0.05). After transfected with lentivirus, there was no statistically significant difference about the expression of FAF1 mRNA in four groups (P> 0.05).5. After infected with Hp culture filtrate, the expression of IKKβ, p65 mRNA and protein in LV-NC-RNAi group and untransfected group were significantly higher than those before Hp infection (P<0.01), but the expression of FAF1 mRNA and protein in LV-NC-RNAi group and untransfected group were significantly lower than those before Hp infection (P< 0.01). There was no statistically significant difference about the expression of IKKβ, p65 and FAF1 mRNA and protein in LV-IKKβ-RNAi group and LV-p65-RNAi group before and after Hp infection (P> 0.05).6. The results of CCK8 cell proliferation experiment showed that, the proliferation of cells in LV-IKKβ-RNAi group and LV-p65-RNAi group increased significantly compared with LV-NC-RNAi group and untransfected group(P< 0.01). There was no statistically significant difference about the proliferation of cells in LV-NC-RNAi group and untransfected group (P> 0.05).7. After infected with Hp culture filtrate, the expression of IKKa/β and p65 protein on NF-κB signaling pathways in LV-IKKβ-RNAi group and LV-p65-RNAi group were significantly lower than that in untransfected group and LV-NC-RNAi group (P< 0.05), and the expression of IκBα protein was significantly higher than that in untransfected group and LV-NC-RNAi group (P < 0.05). The expression of phosphorylated IKKα/β、p65、IκBα(P-IKKa/β, P-p65, P-IκBa) protein on NF-κB signaling pathways in LV-IKKβ-RNAi group and LV-p65-RNAi group were significantly lower than that in untransfected group and LV-NC-RNAi group after Hp infection (P< 0.05). There was no statistically significant difference about the protein level of cells in LV-NC-RNAi group and untransfected group (P> 0.05).8. The concentration of TNF-a and IL-8 in LV-IKKβ-RNAi group and LV-p65-RNAi group were significantly lower than that in untransfected group and LV-NC-RNAi group after Hp culture filtrate infection (P< 0.01). There was no statistically significant difference about the expression of TNF-α and IL-8 in LV-NC-RNAi group and untransfected group (P> 0.05).CONCLUSIONSGene silencing of NF-κB signaling pathways through siRNA lentivirus targeted IKKβ or p65 could affect the cell proliferation of gastric cancer cells overexpressing FAF1, and inhibit down-regulation of FAF1 expression and up-regulation of IKKβ and p65 expression caused by Hp infection, and could inhibit activation of the pathways and related inflammatory reaction regulated by Hp infection in gastric cancer cells overexpressing FAF1. It turned out that Hp infection might regulate FAF1 expression through NF-κB signaling pathway, which might provide new opinion for targeting NF-κB pathway to treat gastric cancer.