Abstract
BackgroundHepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis. However, the underlying mechanism remains elusive. We have reported that HBx is able to up-regulate survivin in hepatocellular carcinoma tissues. The oncopreotein hepatitis B X-interacting protein (HBXIP), a target of miR-520b, is involved in the development of cancer. In this study, we focus on the investigation of hepatocarcinogenesis mediated by HBx.MethodsThe expression of HBx and survivin was examined in the liver tissues of HBx-Tg mice. The effect of HBx/survivin on the growth of LO2-X-S cells was determined by colony formation and transplantation in nude mice. The effect of HBx/survivin on promoter of miR-520b was determined by Western blot analysis, luciferase reporter gene assay, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP), respectively. The expression of HBx, survivin and HBXIP was detected by immunohistochemistry and real-time PCR in clinical HCC tissues, respectively. The DNA demethylation of HBXIP promoter was examined. The functional influence of miR-520b and HBXIP on proliferation of hepatoma cells was analyzed by MTT, colony formation, EdU and transplantation in nude mice in vitro and in vivo.ResultsIn this study, we provided evidence that HBx up-regulated survivin in the liver cancer tissues of HBx-Tg mice aged 18 M. The engineered LO2 cell lines with survivin and/or HBx were successfully established, termed LO2-X-S. MiR-520b was down-regulated in LO2-X-S cells and clinical HCC tissues. Our data revealed that HBx survivin-dependently down-regulated miR-520b through interacting with Sp1 in the cells. HBXIP was highly expressed in LO2-X-S cells, liver cancer tissues of HBx-Tg mice aged 18 M and clinical HCC tissues (75.17%, 112/149). The expression level of HBXIP was positively associated with those of HBx or survivin in clinical HCC tissues. In addition, we showed that HBx survivin-dependently up-regulated HBXIP through inducing demethylation of HBXIP promoter in LO2-X-S cells and clinical HCC tissues. In function, low level miR-520b and high level HBXIP mediated by HBx with partner survivin contributed to the growth of LO2-X-S cells in vitro and in vivo.ConclusionHBx accelerates hepatocarcinogenesis with partner survivin through modulating tumor suppressor miR-520b and oncoprotein HBXIP.
Highlights
Hepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis
We previously reported that HBx was able to up-regulate survivin in hepatoma cells [12]
HBx accelerates hepatocarcinogenesis with partner survivin We have reported that HBx can up-regulate survivin in stable HBx transfected LO2 cells [12], its significance is not clear
Summary
Hepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis. We have reported that HBx is able to up-regulate survivin in hepatocellular carcinoma tissues. The chronic infection of hepatitis B virus (HBV) is a crucial risk factor in the development of HCC. Our laboratory has reported that HBx plays an important role in the event, such as activating Yes-associated protein (YAP), Lin28A/B and Rab18 [4,5,6]. HBx transgenic (Tg) mice are able to develop hepatitis, steatosis, and dysplasia, culminating in the appearance of HCC in liver [7,8,9]. We previously reported that HBx was able to up-regulate survivin in hepatoma cells [12]. HBx may up-regulate survivin through activation of Wnt/β-catenin signaling [13,14,15]. We supposed that HBx might collaborate with survivin to accelerate hepacarcinogenesis
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