Hepatitis B Virus Protein Upregulated HLJ1 Expression Via The Transcription Factor YY1 In Human Hepatocarcinoma Cells

AimTo explore the mechanism of HBV promoting expression of HLJ1 on transcriptional level, to reveal new mechanisms of HBV and hepatocellular carcinoma metastasis, to provide new ideas for the treatment of HCC.Methods1. Real-time PCR and RT-PCR were performed to detect HLJ1 expressions on mRNA level in HepG2 and HepG2.2.15 cell lines.2. pCH-9/3091 was transfected into HepG2 cells at different dose and HLJ1 expressions were detected with real-time PCR and RT-PCR analysis.3. Different lengths of the HLJ1 gene promoter for luciferase assays were generated. HepG2 cells were cotransfected with or without pCMV-SPORT6 or pCH9/3091 plasmid, HLJ1 full-length promoter luciferase reporter constructs, and pRL-TK reporter plasmid. After cultured 48h, luciferase activities were detected. HLJ1 full length promoter luciferase reporter constructs were transfected into HepG2 and HepG2.2.15 cells, luciferase activities were detected.4. HepG2 cells were cotransfected with or without pCMV-SPORT6 or pCH9/3091 plasmid, HLJ1 varying promoter luciferase reporter constructs, and pRL-TK reporter plasmid. After cultured 48h, luciferase activities were detected.5. siRNA plasmids for transcription factor YY1, YY1siRNA-1, YY1siRNA-2 and YY1siRNA-3 were generated. HepG2 cells were cotransfected with pCH9/3091 plasmid, HLJ1 full-length promoter luciferase reporter constructs, siRNA plasmids for transcription factor YY1 and pRL-TK reporter plasmid. After cultured 48h, luciferase activities were detected by using the luciferase assay kit (Dual-Luciferase Reporter Assay System, Promega).6. HepG2.2.15 cells were cotransfected with YY1siRNA-2, then real-time PCR and RT-PCR were performed to detect HLJ1 and YY1 expression on mRNA level.Results1. Real-time PCR and RT-PCR results showed that the amount of HLJ1 expression on mRNA level in HepG2.2.15 cells is higher than that in HepG2 cells.2. HLJ1 in mRNA expression could be upregulated by HBV in a dose-dependent manner as pCH-9/3091 was transfected into HepG2 cells at different dose.3. HLJ1 varying promoter luciferase reporter constructs, pGL3-HLJ1-P, pGL3-F1-P, pGL3-F2-P, pGL3-F3-P were successfully constructed. The relative luciferase activity in HepG2 cells, which were transiently cotransfected with pGL3-HLJ1-P and HBV expression plasmid, is higher than that in control group. The relative luciferase activity of pGL3-HLJ1-P in HepG2.2.15 cells was higher that in HepG2 cells.4. HBV could increase the luciferase activity of the pGL3-HLJ1-P, pGL3-F1-P, pGL3-F2-P constructs, which had the YY1 binding sites, whereas HBV had no effect on the pGL3-F3-P construct which had not the YY1 binding sites.5. When YY1 specific siRNA, pCH9/3091 plasmid and full-length HLJ1 promoter luciferase reporter construct (pGL3-HLJ1-P) were cotransfected into HepG2 cells, YY1siRNA could reduce the activities of HLJ1 promoter in the presence of HBV. When YY1 expression was specifically inhibited by YY1siRNA-2 in HepG2.2.15 cells, HLJ1 expression was also decreased.