The Effects Of Transactivator Of HCV NS5A-NS5ATP9 Gene On The Proliferation Of Hepatoma Cells

HCV is a single-stranded, positive-sense RNA viruses with a genome about 9.4kb in size. Infection with HCV results in chronic hepatitis, cirrhosis and hepatocellular carcinoma. HCV genome only has a long open-reading frame, encoding a polyprotein of about 3010-3033 amino acids that is processed by a combination of host and viral proteases into structural and non-structural protein. More and more results studied on non-structural 5A (NS5A) show that NS5A play an important role in the pathogenesis of HCV.NS5A gene is within Nucleotide positions 6258—7601, encoding NS5A (56 kD) which locates in cytoplasm. NS5A has multiple biological functions. It participates in HCV polyprotein maturation and RNA replication, and serves as a powerful transcription factor to affect transduction pathways of cell signals and activate promoters of genes of many virus and host cells, through which to control and regulate gene expression, cell growth, apoptosis and immune-response. It is closely related with malignant transformation of HCV-infected cells. NS5ATP9 was identified as a NS5A trans-activated protein in suppression subtractive hybridization and play an important role in cell cycle regulation, apoptosis, and the pathogenesis of hepatic fibrosis.To investigate the effects of HCVNS5A on NS5ATP9 expression and NS5ATP9 expression on the proliferation and cycle of HepG2 and HuH-7 cell, in this paper, we used molecular technology to respectively transfect the recombinant plasmid pcDNA3.1-NS5A, pcDNA3.1-NS5ATP9 and vector pcDNA3.1 into both HepG2 and HuH-7 cell lines. The G418-resistant stable clones were attained by screening with G418 for more than two weeks. Compared with stable clones tansfected with pcDNA3.1, stable clones tansfected with pcDNA3.1-NS5A significantly increase NS5ATP9 mRNA with real-time RT-PCR assay, stable clones tansfected with pcDNA3.1-NS5ATP9 significantly increase the percentage of S phase cells of HuH-7 and HepG2 determined by flow cytometry and dramatically stimulate these cells proliferation determined by celltiter-Glo luminescent cell viability assay.These results help further our understanding of interaction between NS5A and NS5ATP9, and consequence affect on cell biology.