MiR-338-3p on HBx Deletion Mutants (HBx-d382) Figure 4. miR-338-3p inhibits cell proliferation by Edu assay. (A–B) Representative profiles of Edu cell proliferation after transfection with miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells compared to their negative control transfection (magnification 1006). (C) Rate of Edu positive cells in S phase. Gain of miR-338-3p inhibits the cellular DNA replication in HBx-expressed LO2 cells and control cells, whereas loss of miR-338-3p expression demonstrates an adverse result, especially in LO2/HBx-d382 cells. (*P,0.001; **P,0.01; ***P,0.05). doi:10.1371/journal.pone.0043204.g004 effect. As the cyclin proteins are known to regulate cell pro- liferation and cell cycle, we tested whether CyclinD1 was differentially expressed in the engineered LO2 cells. PCR and western blot results showed that CyclinD1 mRNA and protein levels, respectively, were up-regulated in cells transfected with HBx and HBx-d382, when compared with those transfected with pcDNA3.0 (Fig. 2); consistent with the data in Figure 1, the effect of HBx-d382 on CyclinD1 expression was more prominent than wild-type HBx. This result indicated that HBx and the HBx-d382 deletion mutant increased CyclinD1 expression in hepatocytes. Relative Expression of miR-338-3p in Normal Hepatocytes is Higher than that in HBx-expressing Cells Our unpublished studies showed that miR-338-3p was down- regulated in HBx-expressing LO2 cells compared with control LO2/pcDNA3.0 cells by microarray and real-time PCR; howev- er, whether miR-338-3p expression was altered in normal hepatocytes compared to HBx-expressing cells was unknown. To address this, real-time PCR evaluation using the 22DDCT method showed that the relative expression of miR-338-3p in normal hepatocytes is higher than that in HBx-expressing cells (Fig. S1). Moreover, according to the results from Figure 2, we conclude that CyclinD1 expression inversely correlates with miR-338-3p expression in HBx-expressing LO2 cells. miR-338-3p Inhibits Cell Proliferation, Especially in LO2/ HBx-d382 Cells We next wanted to determine whether the HBx-regulated miRNA miR-338-3p that we identified in our miRNA microarray could regulate the HBx-mediated increased proliferation we observed in Figure 1. To examine the functional role of miR- 338-3p, we altered cellular miR-338-3p expression by transfecting the HBx- and HBx-d382–expressing LO2 cells with miR-338-3p mimics or an miR-338-3p inhibitor compared with LO2/ pcDNA3.0 cells. Cell cycle and EdU incorporation assays were used to determine cell proliferation. Compared to their respective negative controls (NC), the HBx-expressing LO2 cells transfected with the miR-338-3p mimics displayed significantly higher frequency of cells at the G1 phase and a lower frequency of cells at S phase (Fig. 3A–C, Fig. S2, p,0.001). In contrast, cells transfected with miR-338-3p inhibitor showed a significant de- crease of cells at G1 phase and an increase of cells at S phase (Fig. 3A–C, Fig. S2, p,0.001). This indicated that miR-338-3p inhibited cell cycle progression from the G1 phase to the S phase. Next, we used the EdU incorporation assay, which is a more sensitive and specific method [19–20], to evaluate the effects of miR-338-3p on cell proliferation. We found that the number of cells incorporating EdU in the miR-338-3p mimic-treated group was significantly reduced as compared to the negative control. In contrast, the number of cells incorporating EdU was significantly increased in the miR-338-3p inhibitor-treated group as compared with the negative control. Interestingly, this effect is especially significant in LO2/HBx-d382 cells (Fig. 4A–C). In addition to regulating cell cycle transition between the G1/S phase, this result indicated that miR-338-3p also inhibited cell proliferation, especially in LO2/HBx-d382 cells. Finally, the effects of miR- 338-3p mimics/inhibitors on the colony-forming assay demon- strated that the miR-338-3p inhibited the non-anchored growing ability of transfected LO2 cells, especially in HBx-expressing cells (Fig. 5). CyclinD1 is a Direct Target of miR-338-3p, and the Effect of miR-338-3p on CyclinD1 is Mainly Dependent on the CyclinD1-39-UTR Region (nt 2397–2403) As predicted by several in silico methods for target-gene prediction, including TargetScan [21], CyclinD1 was identified as one of the candidate genes regulated by miR-338-3p. Two putative binding sites, nt 907–913 and nt 2397–2403, are located in the CyclinD1-39-UTR. To determine which putative binding site is used by miR-338-3p, or whether both are used, we mutated each of these putative sites alone (Mut1-CyclinD1-39-UTR [mutated nt 907–913] and Mut2-CyclinD1-39-UTR [mutated nt 2397–2403nt]) as well as both of these sites together (Mut- CyclinD1-39-UTR) in the 39-UTR region of the CyclinD1 gene. WT-CyclinD1-39-UTR was used as a control. PCR amplification was performed using specific primers for the mutated binding sites. The amplified PCR products and genomic DNA isolated from the genome of LO2/HBx cells was used as a PCR template. As shown in Figure 6A, the different CyclinD1-39-UTR gene-derived PCR products were found around the predicted 1573 bp band size. The plasmids were then extracted and subjected to a double digestion with XhoI and NotI enzymes that generated one product: a band at approximately 1573 bp (Fig. 6B) that corresponds to the CyclinD1-39-UTR gene for all plasmids. Finally, the recombinant plasmid gene sequences were further verified by DNA sequencing. To validate whether miR-338-3p can directly regulate Cy- clinD1 through either or both of the putative binding sites in the 39-UTR region of CyclinD1, the WT-, Mut-, Mut1-, and Mut2- CyclinD1-39-UTR plasmids were cloned into the 39-UTR of the Renilla luciferase gene and were co-transfected with miR-338-3p mimics or negative control RNA in LO2/HBx cells. The luciferase enzyme activity levels were then measured to determine the miR- 338-3p effects on luciferase translation upon binding to the WT or mutated 39-UTR regions from CyclinD1. Compared with the negative control group, Renilla luciferase activity was significantly decreased (i.e. the ratio of hRluc to hLuc deceased) in the group co-transfected with miR-338-3p and pCyclinD1-39-UTR-WT (Fig. 6C, p,0.001), as predicted. However, luciferase activity in the group co-transfected with miR-338-3p and pCyclinD1-39- UTR-Mut did not exhibit a significant difference from the NC group (p = 0.404), indicating that miR-338-3p could no longer bind. To determine if one or both of the binding sites were necessary for the functional effect of miR-338-3p, we compared the luciferase activity between pCyclinD1-39-UTR-Mut1 and pCyclinD1-39-UTR-Mut2 groups with the negative control group. The CyclinD1-39-UTR-Mut1 maintained the significant differ- ence (p,0.001) noted in the pCyclinD1-39-UTR-WT in response to miR-338-3p as compared with the negative control group, indicating that miR-338-3p was independent of this binding site. In contrast, the luciferase activity in pCyclinD1-39-UTR-Mut2 group was less affected by miR-338-3p (Fig. 6D, p = 0.04), indicating that this binding site is important for miR-338-3p binding and function. These data indicated that miR-338-3p has effect on both binding sites in CyclinD1-39-UTR region, but that the main binding site is the second site (nt 2397–2403). PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43204