Abstract
ObjectiveHepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.MethodsWe established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.ResultsHBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition.ConclusionmiR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.
Highlights
Among the four open reading frames (ORFs) in the genome of hepatitis B virus (HBV), the Hepatitis B Virus (HBV) X gene (HBx) correlates the most to liver cancer development like hepatocellular carcinoma (HCC)
To study whether and how the HBV X (HBx) deletion mutation affected the growth and proliferation of hepatocytes, we observed the isolated effect of the HBx-d382 HBx deletion mutant, found previously in our studies to be prevalent in HCC patient tumors, on hepatocyte cell proliferation as compared to the wild-type HBx gene
We measured the miR-338-3p expression by qRT-PCR using the 22DDCT method 48 h after transfection and found that there was a significant increase of miR-338-3p expression after knocking-down HBx compared to the negative controls (Fig. S3), while the western blot showed that CyclinD1 protein levels were significantly downregulated after HBx reduction (Fig. S3), Our findings revealed that high HBx expression up-regulated CyclinD1 and down-regulated miR-338-3p, while HBx reduction leads to down-regulated CyclinD1 and up-regulated miR-338-3p expression,indicating that HBx is necessary for cyclinD1 upregulation
Summary
Among the four open reading frames (ORFs) in the genome of hepatitis B virus (HBV), the HBV X gene (HBx) correlates the most to liver cancer development like hepatocellular carcinoma (HCC). Constructs based on either naturally occurring or artificially designed carboxyl-terminal HBx gene deletion mutants were successfully cloned and encoded a carboxy-terminal truncated HBx protein. These HBx mutants have very different functions than wild-type HBx protein [8,9], since the mutants exhibited the ability to promote cell proliferation and to reduce the response to apoptotic stimuli [9,10] that play important roles in HCC development. The mechanism underlying HBx deletion mutant-induced malignant transformation of liver cells is still unclear
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