suggesting that miR-338-3p may play an important role in liver cancer formation due to its location in the fragile site locus associated with liver cancer tumorigenesis. Currently, there are few studies on miR-338-3p, although it was reported that COXIV and SMO could be regulated by miR-338-3p [36–37]. SMO, a transmembrane protein, has a switch function in Sonic hedgehog (Hh) signaling pathway; the classic Hh signaling pathway has been associated with tumorigenesis in the literature [38–39], further implicating a close correlation between miR-338-3p and tumor development. The focus of our study is on CyclinD1, a novel target of miR-338-3p. G1/S-specific CyclinD1 is a protein in the highly conserved Cyclin family and functions at the transition between the G1 and S cell cycle phases where it is a key signaling protein for G1 cell proliferation [40]. CyclinD1 overexpression leads to a remarkable reduction in the lag time between the G1/S phase cell cycle transition as well as promotion of the cell cycle progression rate, eventually leading to uncontrolled cell pro- liferation and tumor formation. In contrast, CyclinD1 inhibition in KM20 cells by effective siRNA treatment leads to cell cycle arrest and cell growth inhibition [41]. Therefore, CyclinD1 can be considered as an oncogene [42–43]. Previous studies have suggested a link between HBx and CyclinD1, where CyclinD1 is shown to be up-regulated by HBx; in these studies, the process is independent of extracellular mitotic signals [44] or is mediated by the NF-kB2(p52)/BCL-3 complex [45]. However, our findings have revealed a new process to explain how HBx can up-regulate CyclinD1, and that mechanism is by down-regulating miR-338- 3p. Therefore, HBx, especially the HBx-deletion mutant HBx- d382, is involved in regulating CyclinD1 expression through an HBx-mediated miR-338-3p down-regulation mechanism, suggest- ing that miR-338-3p plays a tumor suppressor role in the development of HBx- and HBx-d382 deletion-mutant–mediated HCC. The effects of HBx on cell cycle progression are potentially complicated. Most studies found that HBx stimulated cell cycle progression that accelerated transit through checkpoint controls at G0/G1 [44,46–47], which is consistent with our studies. However, several other studies show that HBx actually inhibited cell proliferation by using Hepa1–6 (mouse HCC), HepG2 (human HCC) [48], or hepatocytes isolated from HBx-expressing mice [49]. One possible reason for the contradictory results may be the use of different cell lines with different differentiation statuses and different genetic backgrounds. Another possibility is that the HCC tumor cells may modify the biological activity of HBx, which is something we tried to address in our present study using normal hepatocytes. Yet another possibility is that p53 levels may influence HBx function, as p53 is known interact with HBx, and the normal physiological level of p53 displays anti-apoptotic activity, whereas a high level of p53 displays an adverse result [50– 51]. The recent studies on HBV-related HCC found that genetic heterogeneity of the HBx gene is prevalent in liver cancer tissue. For example, wild-type HBx and mutant HBx can coexist in the same tumor [10]. The major mechanism underlying the creation of HBx deletion mutants is loss of C-terminal fragments of different sizes; this is a dominant phenotype that accounts for almost 100% of the HBx present in the integrated HBV DNA [8,10,52]. The HBx protein functional domains are divided into the regulatory region and the trans-activation region, where the regulatory region is located near the N-terminal end (1,50 aa) and the trans-activation region is located near the carboxyl terminus (51,154 aa) end. The 1,20 aa in the regulatory region is responsible for inhibiting the activity of the trans-activation region [53]. Therefore, a point mutation or deletion mutation MiR-338-3p on HBx Deletion Mutants (HBx-d382) occurring at almost any region in the HBx gene could affect its trans-activation function. Since the deleted region of the HBx- d382 (nt 382–400 deletion) mutant is located near the C-terminal trans-activation region and is also close to where the trans- activation domain associates with mitochondria in HBV-infected cells (the first l32,139 amino acids), the deleted region is necessary for HBx trans-activation activity as well as its known association with mitochondria. Therefore, the HBx-d382 mutant has a close correlation with the process of HBx gene replication and transcription, which are involved in the viral-host interaction, as well as the apoptosis machinery located in the mitochodria. While the loss of C-terminal expression in HBx-d382 mutant attenuates the effect of wild-type HBx on cell proliferation, the expressed truncated HBx protein has gained the ability to promote the malignant transformation of cells. In addition to the role described here, the deleted region in the HBx-d382 mutant is also known to be involved in the p53-binding region of the HBx gene (102,136 aa). Direct binding of HBx to human tumor suppressor p53 has been reported to inhibit p53-stimulated transcription; additionally, the p53 and HBx interaction also reduced the HBx- mediated trans-activation [54]. Therefore, loss of the p53 binding site in HBx mutants can affect the tumor-suppression ability of p53 and further promote tumor formation. Taken together, these results collectively suggest that multiple HBx gene mutations found in HCC, including HBx-d382, may promote the development of HBx-mediated liver cancer. In conclusion, our study revealed that the HBx-d382 mutant has an inhibitory effect on miR-338-3p expression that is associated with HBx-d382-mediated promotion of liver cell proliferation. The finding that HBx-d382 attenuated the miR- 338-3p-mediated inhibition of CyclinD1 expression may provide insight into HBx-mediated tumorigenesis and a new direction for further investigation into HBx function and therapeutic de- velopment. Supporting Information Figure S1 miR-338-3p expression in normal hepato- cytes of LO2 and QSG7701 cells compared with HBx- expressing cells by real-time PCR. (A) Relative expression of miR-338-3p in engineered LO2 cells. 1: LO2; 2: LO2/pcDNA3.0; 3: LO2/HBx; 4: LO2/HBx-d382. (B) miR-338-3p expression in QSG7701 cells transfected with HBx using real-time PCR. 1: QSG7701; 2: QSG/pcDNA; 3: QSG/HBx; 4: QSG/HBx-d382 (*p.0.05, **p,0.001). (TIF) Figure S2 Cell cycle analysis of control LO2/ pcDNA3.0 cells. Histograms of average of G1/S phase popula- tions in LO2/pcDNA3.0 cells after transfection with miR-338-3p mimics or inhibitor (*P,0.001, **P,0.01). (TIF) Figure S3 miR-338-3p and CyclinD1 expression after knocking-down HBx in HBx-expressing cells. (A–B) HBx mRNA and protein expression levels after transfection with HBx siRNA compared with control RNA in LO2/HBx and LO2/ HBx-d382 cells (*P,0.001). (C) Relative expression of miR-338- 8p using the 22DDCT method of qRT-PCR after altering HBx in LO2/HBx and LO2/HBx-d382 cells (*P = 0.013,**P = 0.008). (D) CyclinD1 levels in HBx-expressing cells after transfection of the HBx siRNA or negative control (***P,0.001). Data are shown as mean 6SD of 3 separate experiments. (TIF) PLOS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e43204