MiR-338-3p on HBx Deletion Mutants (HBx-d382) expressing cells was significantly higher than in control LO2 and LO2/pcDNA3.0 cells (*P,0.01). While within the groups of LO2/HBx-d382 LO2/HBx, the former was higher than the latter (#P,0.01). doi:10.1371/journal.pone.0043204.g001 GAAGTGCGAGGAGG-39 (forward) and 59-GGATG- GAGTTGTCGGTGTAGATG-39 (reverse); for GAPDH, 59- CGGATTTGGTCGTATTGGGC-39 (forward) and 59- CCTGGAAGATGGTGATGG. GATT-39 (reverse). qRT-PCR was performed on an Applied Biosystems 7500 RT-PCR System. The cycling conditions for amplification were as follows: 95uC for 1 min, 40 cycles of 95uC for 15 sec, and 58uC for 35 sec. The data were normalized against GAPDH. Each sample was analyzed in triplicate, and the fluorescence signal was measured at each extension step. The relative expression was determined using the 22DDCT method, where the normalized CT (DCT) value was calculated by subtracting the CT of a control gene (U6 snRNA for miR-338- 3p and GAPDH for CyclinD1) from the CT of the gene of interest. Western Blot Analysis HBx and CyclinD1 proteins were measured by western blot. Total protein was extracted from transfected cells using the RIPA lysis buffer (Beyotime, China) according to the manufacturer’s instructions. For western blot analysis, equal amounts of protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Blots were blocked using with 5% skim milk, followed by incubation with antibodies specific for rabbit anti-HBx (Abcam, England, 1:1500 dilution) or mouse anti-CyclinD1 (Santa Cruz, USA, 1:100 dilution), and mouse anti- b-actin (Abcam, 1:3000 dilution). Blots were then incubated with goat anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch, USA, 1:5000 dilution) and visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences, USA). Gene Sequence Analysis and Primer Design Through in silico miRNA target prediction, we found two binding sites for miR-338-3p in the 39 untranslated regions (39- UTR) of CyclinD1. Two gene fragments corresponding to the two binding sites in CyclinD1-39-UTR were cloned into a vector using the restriction enzymes XhoI and NotI. Using the NM_053056 (gene = ‘‘CCND1’’) gene sequence, primers were designed to amplify binding site locations in the 39-UTR region. Mutation primers mutating the specific binding sequence for miR-338-3p in CyclinD1 were also designed, named Mut (mutating both binding sites), Mut-1 (mutating nt 907–913), or Mut-2 (mutated nt 2397– 2403). The PCR products were cloned into a pmirGLO Vector (Promega, Madison, WI, USA) that was designed to quantitatively evaluate miRNA binding and function by measuring luciferase activity after inserting miRNA target sites downstream or 39 of the luciferase gene. In this vector, Renilla luciferase was used as the primary reporter, while firefly luciferase acts as a control reporter for normalization and selection. PCR products were 1573 bp in size. The primers used for cloning were as follows: Wt-Forward: 59- CCGCTCGAGTC CTATTTTTGTAGTGACCTGTTTATG- 39, Wt-Reverse: 59- GAATGCGGCCGCGC TA CGCCCCCGATCAGATGAAG-39; Mutant-Forward: 59- CCGCTCGAGTCC TAT TTTTGTAGTGACCTGTTTAT- GAGTTCCAGATTTTCTACCCAACGGCCC-39, Mutant- Reverse: 59-GAATGCGGCCGCGCTACGCCCCCGATCA- GATGAAGT. GCTCTGGAACACAGGCGCAGGGAAGAGAAG-39; Mu- tant1-Forward: 59-CCGC TCGAGTCCTATTTTTGTAGTGACCTGTTTAT- GAGTTCCAGATTTTCTACCCAACGGCCC-39, Mutant1- Reverse: 59-GAATGCGGCCGCGCTACGCCCCCGATCA- GAT GAAG-39; Mutant2-Forward: 59-CCGCTCGAGTCC- TATTTTTGTAGTGACCTGT TTATG-39, Mutant2-Reverse: 59-GAATGCGGCCGCGC- TACGCCCCCGATCAGATG Figure 2. HBx, especially HBx-d382, enhances CyclinD1 expression. (A) CyclinD1 protein level of the engineered LO2 cells. (B) The histogram of CyclinD1 mRNA and protein level in LO2/HBx and LO2/HBx-d382 cells, compared with LO2/pcDNA3.0 cells (*P,0.001,**P = 0.017,***P = 0.029). doi:10.1371/journal.pone.0043204.g002 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43204