Cross-talk Between Notch And NF-κB Pathways In The Development Of Hepatitis B Virus X Protein Associated Hepatocellular Carcinoma

Objective1. To establish the HBx -transfected L02 cell model L02/HBx .2. To observe the binding ability of HBx with Notch and NF-κB in the HBx -transfected cell strain L02/HBx , investigate the potential relationship of the two pathways with HBx involved in the development of hepatocellular carcinoma and explore the mechanisms of hepatocellular carcinoma from the new signaling pathways.3. To observe the expression of the subunits of NF-κB p50,p65 and inhibitor IκBαmRNA and protein, as well as the change of NF-κB DNA binding activity in L02/HBx cells after blocking Notch signal pathways, and explore the cross-talk between Notch and NF-κB pathways in the development of HBx- associated hepatocellular carcinoma .Methods1. The HBx-transfected cell strain L02/HBx was been successfully established previously. The L02/HBx cells were cultured.2. Coimmunoprecipitation assay was applied to detect the physical interactions of Notch1 (NICD, Jagged1) and NF-κB (p50, p65, IκBα) with HBx , and immunofluorescence assay was performed to investigate the co-localization of HBx with NICD in the L02/HBx cells. 3. To treat the L02/HBx cells with 20μM DAPT (DAPT group) or 0.05%DMSO (DMSO group) for 48h, western blot was performed to detect the expression of NICD in L02/HBx +DAPT, L02/HBx+DMSO and L02/HBx cells.4. Electrophoretic mobility shift assay (EMSA) was used to observe the NF-κB DNA binding activity in DAPT group, DMSO group and L02/HBx cells.5. Real-time quantitantive polymerase chain reaction (Real-time qPCR) was employed to examine the expression of NF-κB (p50, p65, IκBα) mRNA in the DAPT group, DMSO group and L02/HBx cells. And western blot was performed to detect the protein levels of p50, p65 in cytoplasim and nucleus respectively, and the total protein level of IκBαin DAPT group, DMSO group and L02/HBx cells.Results1. The results of coimmunoprecipitation and immunofluorescence assay demonstrated that NICD was co-localizated and coimmunorecipitated with HBx, and no specific interaction was found between Jagged1, p65, p50 or IκBαwith HBx in the L02/HBx cells.2. Our Western blot result showed that the expression of NICD in the L02/HBx cells with DAPT was significantly lower than the control cells.3. The results of EMSA showed that the NF-κB DNA-binding activity was inhibited in L02/HBx cells treated with DAPT compared to control cells.4. The Real-time qPCR showed that the expression level of p65 mRNA in the DAPT group(0.466667±0.027285) was significantly lower than that in DMSO group(0.97±0.045092)and L02/HBx(1±0.049329)cells (p<0.01);the expression level of p50 in the DAPT group(0.43±0.01)was significantly lower than that in DMSO group(1.01±0.01)and L02/HBx(1±0.04)cells (p<0.01);In contrast, the expression of IκBαmRNA in the DAPT group(1.38±0.025166) was higher than that in DMSO group(1.006667±0.012019)and L02/HBx(0.995±0.095)cells (p<0.01). 5. The results of Western blot demonstrated that the total protein level of IκBαwas increasd, the nuclear extracts levels of p65 and p50 were decreased in L02/HBx cells treated with DAPT compared to control cells, and no significant change was observed in the cytoplasmic protein of p65 and p50 in L02/HBx cells treated with or without DAPT.ConclusionsHBx could induce Notch signaling pathway by binding to NICD, and the activation of Notch signaling up-regulates the expression of p50 and p65; down-regulates the expression of the suppressor IκBα; stimulates the nuclear transport of NF-κB dimers and the NF-κB DNA-binding activity, resulting in the activation of NF-κB pathway and thus to regulate target genes. Therefor, HBx promotes the development of hepatocellular carcinoma via regulating Notch signaling directly and NF-κB by Notch pathway.