HBV Regulated Raf1 Expression In HEPG2.2.15 Cells Through Enhancing Its Promoter Activity

ObjectiveTo investigate the effects of hepatitis B virus on the expression of Raf1, to reveal its regulatory mechanism, and provide useful information for understanding mechanism of HCC induced by HBV infection in a new insight.Methods1. RT-PCR and Western blot were performed to detect the expression of Raf1 in HepG2 and HepG2.2.15 cells.2. The Raf1 promoter luciferase reporter plasmid (pGL3-Basic-Raf1-P) was successfully constructed. pGL3-Basic-Raf1-P was transfected into HepG2 cells to detect its activity. pGL3-Basic-Raf1-P and HBV expression plasmids or control plasmids were transiently co-transfected into HepG2 cells, respectively. Then their promoter activities were detected.3. pGL3-Basic-Raf1-P and HBs, HBp, HBc or HBx expression plasmids were transiently co-transfected into HepG2 cells, respectively. Then their promoter activities were detected. To further determine the phenomenon, Western blot was performed to detect the effects of these four proteins on the expression of Raf1.4. siRNA plasmids were generated to interfer the HBV expression. RT-PCR was used to detect the interference effect, and Western blot was performed to detect the expression of Raf1 after siRNA plasmids transfected into HepG2.2.15 cell.Results1. Raf1 relative expression increased 2.16 times in the presence of HBV than that in absence of HBV with RT-PCR analysis. Meanwhile, the expressions of Raf1 protein were obviously stronger in HepG2.2.15 cells than that in HepG2 cells when analyzed with Western blot.2. pGL3-Basic-Raf1-P had strongly activity compared with the control group. HBV could increase Raf1 promoter activity 4.65 times as the control group.3. HBs and HBx could significantly enhance the Raf1 promoter activities 3.62 and 2.92 times as the control group, respectively. However, HBc and HBp had little effect on the expression of Raf1. Meanwhile, Western blot analysis also showed that HBs and HBx could significantly increase the Raf1 protein expression.4. When the siRNAs were transfected into HepG2.2.15 cells, relatively, the expression of HBs and HBx were inhibited. Meanwhile, the expressions of Raf1 were decreased.ConclusionWe showed that HBV could up-regulate the Raf1 expression in HepG2.2.15 cells through enhancing its promoter activity, and HBs, HBx proteins might play major roles in the process. This study reveals a new pathway of HBV causing HCC, provides a new mentality to therapy the HBV-related HCC.