RNA Interference To Knock Down Hepatitis B Virus X Gene Expression On The Experimental Therapy For Hepatocellular Carcinoma

Background Hepatocellular carcinoma (HCC) has been ranking the third leading patients suffering from cancer to death. With the great progression of treatments for HCC, most of the HCC patients shall die off, however, in about 6 months for the fast growth of HCC. It is critical for us to find more potential therapy for it. Whereas, the emergence of gene therapy brings hope for cure it thoroughly.More than 90% of HCC patients are positive for hepatitis B virus surface antigen in sera. Therefore, HCC is closely related to hepatitis B virus. The specific hepatitis B virus X protein (HBx protein) had been identified in more than 80% of the liver tissue from HCC patients. It had been proved sufficiently that HBx protein was tied up closely with the growth and development of HCC. Hence, the HBx protein should have been the good target for the gene therapy of HCC.RNA interference is to knock down mRNA specifically, which expressed by suited gene by double strands RNA, which is gene silencing of post transcription. RNA inference is a kind of economical, convenient, high potent tool to inhibit gene expression, and might be a new way to determine the gene function and for the gene therapy.Objective To knock down HBx gene expression by shRNA on the experimental gene therapy for HCC. Methods The HBx gene cloning procedures were performed on gemone DNA of MHCC97-H, the HBV DNA integrated and HBsAg negative human HCC cell line. The PCR products were sequenced before BLAST. With HBx full-length gene cloning, it was determined that the relation between the HBV DNA integration site and the terminal of HBx gene open reading frame primarily. The transcription of HBx gene was approved by RT-PCR and the expression of HBx protein by immunoflourescence. Three shRNA sequences, two targeting the cloned HBx gene named X164(nt 164-182), X215(nt 215-233) and another control sequence named MOCK, were synthesized, which were cloned into pGenesil-1 vector. After confirmed by restriction enzyme reaction, PCR assay, sequencing, MHCC97-H cell line were instant transfected with the shRNA vectors by lipofectamine 2000. The transfection efficiencies were analyzed by FACS. Three shRNA vectors were transferred into MHCC97-H cells by electroporation. The stable shRNA expression cell clones were selected by G418 and picked out at 30-35d after transfection. The RNAi efficiency to HBx gene transcription and the level of HBx protein in these clones were tested by relative quantity with RT-PCR and cell immunofluorecence staining respectively. The biological characteristics of the target clones selected by in vitro and in vivo screening were studied. Results It was demonstrated that 247bp fragment of HBx gene be cloned from the genome DNA of MHCC97-H after sequencing, which must have been adr subtype and C genotype by BLAST. It was speculated that HBx gene was the 3' terminal deleted, where might have been the integration site. With strict control scheme, the HBx mRNA and HBx protein in MHCC97-H were confirmed respectively. The pshRNA- X164/X215/MOCK vectors were constructed successfully by confirming procedures. With transient transfection, EGFP expressed by shRNA plasmids gave out green light in MHCC97-H cells. The transfection efficiency of pshRNA-X164/X215/MOCK was 40.25±3.2%, 25.29±0.52%, 50.60±0.99% respectively. Demise MHCC97-H cells after pshRNA-X215 transfection were more than that of pshRNA-X164/MOCK. The clone numbers of 215 picked out were less than MOCK and 164 obviously. The shRNA specific to HBx (X215) could knock down the HBx mRNA by 70-93% in 6 of 215 cell clones successfully, and reduce the protein level as well. RNAi targeting HBx expression in MHCC97-H cells confirmed evident delay in G0/G1 of cell cycle, reduction in cell proliferation and tumor growth in nude mice respectively, but not eradicating and inhibiting the growth of MHCC97-H cells completely. Conclusion The HBV DNA integrated and HBsAg negative MHCC97-H is the HBx gene selectively expressed human HCC cell line. shRNA vectors specific to cloned HBx gene were constructed successfully, which expressed in MHCC97-H cell line by instant transfection. Knocking down the HBx expression by RNAi could resolve the difficulty of therapy for HCC partially, which suggest that HBx protein plays an important role in maintaining HCC growth.