Preliminary Study And Screening Of The Up- And Down-regulation Genes Correlated With HBx COOH-terminally Deleted 40 Amino Acids

Chronic infection of hepatitis B virus (HBV) is the leading cause of the hepatocellular carcinoma (HCC) in worldwide. The viral transactivator HBV X (HBx) gene has been closely associated with the molecular pathogenesis of HBV-related HCC. Natural deletions of HBx protein is characterized as a common feature in tumor tissue from the HBV-related HCC case. The mutant of HBx gene may plays a critical role in the hepatocarcinogenesis. Previous study showed that the HBx COOH-terminally deleted 40 amino acids plays a key role in controlling cell viability and proliferation in Huh7 cell line in vitro. To further explore the impact of the HBx mutant COOH-terminally deleted 40 amino acids on the HBx-mediated hepatocarcinogenesis, The up and down regulation differential expression genes were screened using suppression subtractive hybridization (SSH) technique and cDNA microarray in Huh7 cells transfected with pcDNA-HBx or pcDNA-HBx3'-40. The 87 differentially expressed genes were identified by SSH, including 55 up regulated and 32 down regulated genes;and 167 differentially expressed genes were identified by cDNA microarray including 145 up regulated and 22 down regulated genes. Some clones were analyzed by sequencing and bioinformatics for potential function and possible relationship with hepatocarcinogenesis. Three significant genes, Wnt5a, ETV5, DKFZ, were further explored. The mRNA expression of these three genes in hepatocellular carcinoma tissues were investigated by the reverse transcription polymerase chain reaction (RT -PCR) and real-time flurogenic quantitative RT-PCR (RQ-PCR). The results showed that expression levels of the three mRNAs in the Huh7 cells transfected with pcDNA-HBx3'-40 were higher than that in the Huh7 cells transfected with pcDNA-HBx, the same results were obtained in the adjacent tissues of hepatoma compared with the hepatoma tissues except the Wnt5a gene. It means that the two genes, ETV5 and DKFZ, may be involved in the cell proliferation. The expression of Wnt5a , P21^WAF1, P53 proteinwere detected by immunohistochemistry and western blot in the hepatoma and adjacent tissues. The results showed that the expression of P53 and P2rAM in the hepatoma tissues was higher than that in the adjacent tissues of hepatoma. While the lower expression of Wnt5a was observed in the adjacent tissues of hepatoma in comparison with the hepatoma tissues. The expression of Wnt5a in the Huh7 cells transfected with pcDNA-HBx3'-40 were higher than that in the Huh7 cells transfected with pcDNA-HBx0 While the expression of P21WAFL was reverse. (P<0.01;P<0.05), No significant difference of the P53 expression was found between two cell lines. The expression of Wnt5a is significantly correlated with the expression of P53 and P21WAFl. Wnt5a may be involved in the process of hepatocarcinogenesis by interacting with P53 and P21MF1 signaling molecules.