The effect of miR-338-3p on HBx deletion-mutant (HBx-d382) mediated liver-cell proliferation through CyclinD1 regulation.
OBJECTIVE
Hepatitis 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.


METHODS
We 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.


RESULTS
HBx-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.


CONCLUSIONS
miR-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.
Department of Infectious Disease, Xiangya Hospital, Central South University, Changsha, Hunan, China.
Xiaoyu
X
Fu
Deming
D
Tan
Zhouhua
Z
Hou
Zhiliang
Z
Hu
Guozhen
G
Liu
Yi
Y
Ouyang
Fei
F
Liu
D015153:Blotting, Western; D049109:Cell Proliferation; D003114:Colony-Forming Units Assay; D019938:Cyclin D1 / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008055:Lipids; D008113:Liver Neoplasms / Q000821:virology*; D008156:Luciferases; D035683:MicroRNAs / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
C530104:CCND1 protein, human; D017931:DNA Primers; D008055:Lipids; C086724:Lipofectamine; C532050:MIRN338 microRNA, human; D035683:MicroRNAs; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D019938:Cyclin D1; D008156:Luciferases