Research On Liver Cells Activity And Proliferation Influenced By Up/Down Regulation Of NIRF Gene Expression

Objective: As NIRF protein has the function of E3 ubiquitin ligase, and in our preliminary research we found that NIRF can participate in the ubiquitination degradation process of HBV core protein (HBc) in vitro. In order to have a further understanding of the role of NIRF gene in hepatitis B process caused by HBV infection, we altered NIRF gene expression by up/down regulation in L02 human normal liver cells, HepG2 human hepatoma cells and HepG2.2.15 (2215) cells.Methods:1 Western Blot was carried out to detect NIRF protein expression level in L02, HepG2 and 2215 cells.2 Immunocytochemical methods were performed to detect the sub-cellular localization of NIRF and HBc protein in 2215 cells.3 Construct NIRF-full length eukaryotic expression plasmid and three NIRF targeted RNA interference vectors: Ri-1, Ri-2, Ri-3 and positive control (PC). Select the most effective RNA interference carrier by RT-PCR and Western Blot.4 Transfect the NIRF full-length and interference carrier with corresponding empty vectors into above liver cells by lipofectamineTM 2000, observe the influence on cell proliferation activity by flow cytometry and MTT analysis. Results:1 Western Blot results showed that the NIRF protein expression quantity is highest in 2215 cells, next in L02 cells, least in HepG2 cells; the difference was statistically significant (P < 0.01).2 Most of NIRF and HBc protein sub-cellular localization in 2215 cells showed with a similar distribution scattering in the cytoplasm, and the rest displayed in a gathered granular in the nuclei. The merged image showed HBc and NIRF protein have the same localization in cells.3 Successfully constructed 3 shRNA interference plasmids. Transfected these plasmids into 2215 cells, and detected the mRNA and protein level through RT-PCR and Western Blot analysis. The results showed that the Ri-2 plasmid has the most interference effects.4 The cell cycle distribution showed an induced arrest at G1 phase by over-expression of NIRF in L02 and 2215 cells, while HepG2 cells were affected not apparently. In contrast, the low-expression of NIRF effects on L02 and 2215 cells were not obvious, but it was revealed to accelerate G1 phase transition to S phase in HepG2 cells. MTT analysis presented that as time goes on, over-expression of NIRF suppressed L02 and HepG2 cells'proliferation significantly (P<0.01), low-expression had no such effects; while the performances were completely opposite in 2215 cells.Conclusion: NIRF is highly expressed in 2215 cells, and has directly relation with HBc. Up/down regulation of NIRF gene led to a different reaction in L02 and HepG2 cells compared with 2215 cells. Accordingly, we can speculate that NIRF gene participate in the life cycle regulation of HBV in host cells. And more research is needed to explore the biological function of NIRF in the future.