Effect Of Hepatitis B Virus X Protein On The Proliferation Of Hepatoma Cells Via SPHK1

Hepatocellular carcinoma (HCC) is a common malignancy tumor in clinical and it seriously ruins health of sufferers. Chronic infection with hepatitis B virus (HBV) is one of the major reasons leading to hepatocarcinogenesis. HBV X protein (HBx) encoded by HBx gene of HBV genome has been proven to play a crucial role in HCC development progress, including cell proliferation, migration and invasion. Malignancy tumors are closely associated with cell metabolic disorders. Sphingolipid metabolism is a significant part of lipid metabolism, disorder of sphingolipid metabolism induces tumors and atherosclerosis. Sphingosine kinase 1(SPHK1), the limited enzyme in sphingolipid metabolism, could effectively produce sphingosine 1-phosphate (SIP) which is a vital effector molecule involved in a variety of cellular functions. Studies indicate that SIP level is majorly controlled by SPHK1 related to excessive proliferation of tumor cells. The underlying mechanism by which HBx promotes development of HCC is not entirely clear. In order to elucidate the molecular mechanism by which HBx promotes hepatocarcinogenesis, we investigated the role of SPHK1 in HBx-enhanced hepatocarcinogenesis. This research contains three parts as follows:Part 1:Effect of HBx on the expression of SPHK1To investigate the relationship between HBx and SPHK1, we analyzed 38 paired clinical HCC tissues and para-carcinoma tissues at mRNA level, the data showed that HBx was positive associated with SPHK1. Then, we detected the expression of SPHK1 in the liver or HCC tissues from HBx-Transgenic (HBx-Tg) mice; we found that SPHK1 was up-regulated in 6 months (or 18 months) HBx-Tg mice. Further, we analyzed SPHK1 expression of HepG2 (or LO2) cells which were transiently transfected with pcDNA3.1-HBx and HepG2-X (or HepG2.2.15) cells which were stably express HBx by RT-PCR and Western blot. The results indicated that HBx up-regulates the mRNA and protein of SPHK1 in a dose-dependent manner. On the contrary, psi-HBx down-regulates the mRNA and protein of SPHK1 in a dose-dependent manner. Thus, we conclude that HBx up-regulates the expression of SPHK1 in hepatoma cells.Part 2:Effect of HBx on SPHK1 promoterIn order to clarify the mechanism by which HBx up-regulates the expression of SPHK1, we focused on investigation of the effect on SPHK1 promoter by HBx. Dual-luciferase reporter gene assay revealed that HBx enhanced the activities of SPHK1 promoter, while HBx RNA interfering decreased the activities of SPHK1 promoter. We found an AP2a binding site in the region -20 to-15 of SPHK1 promoter using bioinformatic tools, and then designed primers and constructed pGL3-Basic recombinant plasmid, termed pGL3-320-mut, with deletion of the AP2a binding site. Dual-luciferase reporter gene assay manifested that the relative luciferase activities of pGL3-320-mut were obviously decreased compared to those of pGL3-320, suggesting that AP2a is important for the transcription of SPHK1. ChIP assay further proved that AP2a is able to interact with the SPHK1 promoter. Meanwhile, AP2a siRNA pool significantly abolished the activation of SPHK1 promoter increased by HBx and SPHK1 expression reduced in a dose-dependent manner, suggesting that AP2a is involved in the activation of SPHK1 promoter reinforced by HBx. We detected the expression of AP2a in HepG2, HepG2-X and HepG2.2.15 cell lines, and confirmed that HBx could up-regulate the expression of AP2a. Taken together, we conclude that HBx up-regulates the expression of SPHK1 through transcription factor AP2a.Part 3:HBx promotes cell proliferation through SPHK1We conducted a series of tests to evaluate the role of SPHK1 in promoting proliferation of hepatoma cells mediated by HBx. HepG2-X (or LO2-X) cells were transfected with si-SPHK1; MTT assay and colony formation assay showed that HBx accelerated the proliferation of hepatoma cells, but SPHK1 siRNA blocked the promotion of cell proliferation mediated by HBx, indicating that SPHK1 is involved in the HBx-enhanced proliferation of hepatoma cells. We found that HBx strengthened the growth of tumor transplanted in nude mice, but the treatment with si-SPHK1 could apparently block the tumor growth in nude mice. In addition, immunohistochemistry staining of Ki-67, a marker of proliferation, further attested that SPHKl interfering remarkably blocked the HBx-enhanced tumor growth, suggesting that SPHK1 plays crucial roles in the tumor growth mediated by HBx. Taken together, we conclude that HBx promotes hepatoma growth through SPHKl.In summary, in this study we report that HBx is able to elevate the expression of SPHKl in hepatoma cells through up-regulating transcription factor AP2a, resulting in promoting proliferation of hepatoma cells. Our finding provides new insights into the mechanism by which HBx enhances proliferation of hepatoma cells and new theoretical reference for prevention, diagnosis and therapy of HCC.