The Effect Of HBx On Expression Of DNMT3A And DNMT3B

OBJECTIVE In this study, immortalized human-derived non-tumor liver cell line QSG7701 was used as target cells, recombinant plasmid HBV-X was stably transfected into QSG7701 cells, and observed the expression of DNMT3A/3B in QSG7701 cells.METHODS QSG7701 cells were transfected with the recombination plasmid pcDNA-X or control pcDNA3.0 plasmid using transfection Reagent. Selected medium containing G418 were used to select the stably transfected cell clones. The expression of HBV X mRNA in the HBV-X stably transfected QSG7701 cells was detected by semi-quantitative reverse-transcribe polymerase chain reaction (RT-PCR), and the expression of HBx protein was confirmed by Western blot. The expression of DNMT3A/3B mRNA was detected by Real time quantitative PCR in recombinant plasmid transfected cells (pcDNA-X/QSG7701), empty vector transfected cells (pcDNA3.0/ QSG7701) and untransfected QSG7701 cells. The expression of DNMT3A/3B protein in recombinant plasmid transfected cells (pcDNA-X/QSG7701), empty vector transfected cells (pcDNA3.0/ QSG7701) and untransfected QSG7701 cells was detected by Immunocytochemistry.RESULT RT-PCR analysis and Western blotting showed that HBV X mRNA and HBx protein was expressed stably in pcDNA-X/QSG7701 cell line. Real time Quantitative PCR analysis showed that, the expression of DNMT3A and DNMT 3B mRNA expression in pcDNA-X/QSG7701 cells was higher than which in pcDNA3.0/QSG7701 cells and untransfected QSG7701 cells. Immunocytochemistry analysis showed that:the expression of DNMT3A and DNMT 3B protein in pcDNA-X/ QSG7701 cells was higher than which in pcDNA3.0/QSG7701 cells and untransfected QSG7701 cells.CONCLUSION We established QSG7701 cells line stably transfected with HBV X gene, Which might be a valuable cell model for studying the roles of HBV X gene and HBx protein in hepatitis B virus life cycle and development of HBV related HCC in vitro. The result showed that HBX up-regulate the expression of DNMT3A/3B mRNA and protein in transfected QSG7701 cells, which might affect the expression of some oncogene and anti-oncogene in cells and result in malignant transformation of the cells.