MiR-338-3p on HBx Deletion Mutants (HBx-d382) transfected with miR-338-3p mimics or inhibitor or their respective controls(wP = 0.164,ww P = 0.438, #P = 0.220,##P = 0.101,* P = 0.254,** P = 0.417). doi:10.1371/journal.pone.0043204.g006 miR-338-3p Represses CyclinD1 Protein Expression To directly test the validity of the putative target, HBx- expressing LO2 cells were transfected with miR-338-3p mimics or miR-338-3p inhibitor as well their respective negative controls. The mRNA and protein levels of CyclinD1 were measured by qRT-PCR and Western blotting, respectively. Relative to the control, miR-338-3p overexpression downregulated endogenous CyclinD1 protein, while miR-338-3p inhibition upregulated the CyclinD1 protein; consistent with our previous data, these changes in CyclinD1 are more prominent in the LO2/HBx-d382 cells (Fig. 6E–G). In contrast, no change of CyclinD1 mRNA level was noted (Fig. 6H), indicating post-transcriptional regulation of CyclinD1 by miR-338-3p. Changes in HBx Expression Affect miR-338-3p and CyclinD1 Expression in Transfected LO2 Cells Since the miR-338-3p and CyclinD1 expression was signifi- cantly changed in HBx-expressing LO2 cells, we wondered whether the reduction of HBx could also lead to differential miR-338-3p and CyclinD1 expression. To address this question, we transfected HBx siRNA or control RNA into LO2/HBx and LO2/HBx-d382 cells to inhibit HBx expression. 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 down- regulated 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. Our data introduces a new mechanism suggesting that HBx up-regulated CyclinD1 through down-regulating miR-338-3p. Discussion Although the integration of HBV DNA into the host genome is not required for viral replication, it is a common phenomenon that HBV genes integrate with host genomic DNA in many liver tissues chronically infected with HBV. This promotes HBV persistent infection [22–23] by providing protection of the viral genome from immunological surveillance and, most importantly, promoting the development of genomic DNA instability that contributes to malignant transformation of liver cells [24–25]. In addition to effecting these changes on the infected host liver cells, the process of HBV DNA integration can induce mutations in the HBV DNA itself. Deletion mutations are frequently found in the integrated HBV DNA, especially in the X gene (including the HBx gene explored here), S gene, and enhancer regions, resulting in the expression of truncated proteins encoded by the mutated genes, such as the truncated HBx protein that can then lead to dysregulation of host cellular genes [9,26]. For the HBx gene, deletions in the C-terminal region of the gene are common after HBV DNA integration. In the context of hepatocarcinogenesis, there is an intriguing correlation between the occurrence of this particular mutation and transformed liver cancer cells, as the level of C-terminal truncated HBx protein is higher in transformed tumor tissues as compared to distal non-tumor tissues in HBV- infected HCC patients [10,26]. Supporting this finding, Iavarone et al. [9] found that a truncated HBx protein detected in HCC tissues derived from an HBx gene deletion that led to a reading frame shift mutation and the emergence of a new STOP codon. Currently, the mechanisms underlying how HBx induces hepatocarcinogenesis are not fully clear. Wang et al. [27] reported that HBx-mediated down-regulation of let-7a expression inhibited cell proliferation; this was the first study to report an interaction between HBx and miRNA. However, the role of miRNA in the relationship between the HBx deletion mutation and hepatocarci- nogeness is largely unexplored. To better understand the mechanism underlying the effect of miRNAs on HBx deletion- mutation–related HCC, we performed the study described here, and our results provide new ideas for the diagnosis and treatment of liver cancer. Our previous studies found that the HBx-d382 mutant was detected in several HCC patients [16], and the ability of HBx-d382 to induce tumor formation was confirmed in a nude mice model [28]. miRNA microarray analysis and confirmatory real-time PCR data showed that HBx-d382 had a stronger ability to down-regulate miR-338-3p expression than wild-type HBx in transfected LO2 hepatocyte cells (unpublished data). To un- derstand what genes targeted by miR-338-3p might function to promote HBx deletion-mutation related liver cancer, we used TargetScan to identify the potential miR-338-3p target gene and found that CyclinD1 was a likely candidate. Furthermore, HBx- d382 was able to promote proliferation and anchor-independent growth of LO2 cells as well as enhance CyclinD1 gene expression. Edu incorporation and cell cycle analysis by flow cytometry to measure cell proliferation demonstrated that ectopic expression of miR-338-3p mimics in both LO2/HBx and LO2/HBx-d382 cells inhibited cell proliferation, whereas repression of endogenous miR-338-3p expression enhanced cell proliferation. Furthermore, the forced expression or down-regulation of miR-338-3p reduced or enhanced, respectively, the protein expression of CyclinD1 in both LO2/HBx and LO2/HBx-d382 cells. In the above experiments, the phenotypes were more profound in LO2/HBx- d382 than in LO2/HBx cells. The dual luciferase reporter assay confirmed that CyclinD1 is a direct target gene of miR-338-3p, and that the main binding site of miR-338-3p in CyclinD1-39- UTR region is located between nt 2397–2403 (the second predicted binding site). Finally, HBx reduction enhanced miR- 338-3p expression and reduced CyclinD1 expression in HBx- expressing cells, supporting that HBx is necessary for cyclinD1 upregulation through miR-338-3p. The miR-338 gene is located on chromosome 17q25 and produces two mature forms (miR-338-3p and miR-338-5p). Our data showed that miR-338-3p expression is down-regulated in inducing tumor formation HBx-expressing cells compared with normal hepatocyte cell line LO2 and QSG7701, similar to the results found in another HCC cell line HepG2.2.15 cells [29]. Moreover, another study reported that down-regulated miR-338 expression, and miR-338-3p in particular, was frequently found in HCC tissues [30], which peripherally supports our data that miR- 338-3p is associated with HCC formation. The miR-338 gene is located within the intron 8 region of the apoptosis -associated tyrosine kinase (AATK) gene [31]. Interestingly, the survivin gene, a new inhibitor of apoptosis protein family, is also located on chromosome 17q25 [32]. Overexpression of survivin gene was found in a wide variety of tumor and transformed cells, including liver cancer [32–34] and cells in chronic HBV infected livers that are deemed close to transformation to tumor cells [32,35], PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e43204