The Effect of miR-338-3p on HBx Deletion-Mutant (HBx- d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation Xiaoyu Fu 1 , Deming Tan1*, Zhouhua Hou 1 , Zhiliang Hu 2 , Guozhen Liu1, Yi Ouyang1, Fei Liu1 1 Department of Infectious Disease, Xiangya Hospital, Central South University, Changsha, Hunan, China, 2 The Second Hospital of Nanjing, Nanjing, Jiangsu, China Abstract 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 CyclinD1-39UTR 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 CyclinD1-39UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition. Conclusion: miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-39UTR 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. Citation: Fu X, Tan D, Hou Z, Hu Z, Liu G, et al. (2012) The Effect of miR-338-3p on HBx Deletion-Mutant (HBx-d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation. PLoS ONE 7(8): e43204. doi:10.1371/journal.pone.0043204 Editor: Birke Bartosch, Inserm, U1052, UMR 5286, France Received February 2, 2012; Accepted July 18, 2012; Published August 17, 2012 Copyright: 2012 Fu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by grants from the National Natural Science Foundationof China (No 30872228). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dmtxy2012@163.com Introduction 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). The HBx protein is a multifunctional regulator that is essential for viral replication and plays an important role in regulating gene transcription, participating in cell signaling, and controlling cell proliferation and apoptosis [1–2]. However, there is controversy surrounding the direct causal effect of HBx on HCC development [3–5]. The integration of the HBx gene into the host genome in hepatocarcinoma tissues, and the gene mutants in HBx that arise due to this integration process, have been reported in many studies. The study conducted by Minenura M et al. [6] revealed a correlative relationship between HBx gene point mutations (at codon 130 [AAG R ATG] and 131 [GTC R ATC]) and liver cancer. Another report by Yeh et al. [7] found that HBx-A31 (containing a mutation at codon 31) was detected more frequently in patients with 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. However, the mechanism underlying HBx deletion mutant-induced malignant transforma- tion of liver cells is still unclear. MicroRNAs (miRNAs) are a class of endogenous, noncoding small RNAs that regulate gene expression at the post-transcrip- tional level through binding the 39-UTR region of the target gene mRNA. Several recent studies have shown that dysregulation of PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43204