MiR-338-3p on HBx Deletion Mutants (HBx-d382) Figure 3. miR-338-3p inhibits cell proliferation, especially in LO2/HBx-d382 cells. (A) A representative cell cycle profile for miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells comparing to their control RNA transfection. (B) Average of G1 phase populations in HBx- expressed LO2 cells after transfected miR-338-3p mimics or inhibitor (*P,0.001). (C) Average of S phase populations after transfected miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells (*P,0.001). While miR-338-3p overexpresses or suppresses, miR-338-3p induce cell cycle arrest and anti-miR-338-3p increases cell growth, especially in LO2/HBx-d382 cells. Values in (B) and (C) are means 6SD of three separate experiments. doi:10.1371/journal.pone.0043204.g003 AAGTGCTCTGGAACACAGGCGCAGGGAAGAGAAG- 39. Amplification and Identification of CyclinD1-39-UTR Constructs Amplification of wild-type and mutant plasmids was carried out using the same conditions. Briefly, 6 mL of 56 PrimeSTAR buffer (TaKaRa, Japan), 2 mL of 2.5 mM dNTPmix, 1 mL each of upstream and downstream primer (10 mM), 0.3 mL of PrimeS- TAR HS DNA polymerase (2.5 U/mL), 1 mL of DNA template, and sterile water up to a total of 30 mL per reaction were mixed together. Touchdown PCR was used for amplification as follows: hot-start at 95uC for 5 min, followed by 10 cycles of denaturation at 98uC for 10 sec, annealing starting from 65uC and decreasing 1uC in each cycle, and extension at 72uC for 1 min 45 sec; for the remaining 18 cycles, the annealing temperature was kept at 55uC. A final extension step at 72uC for 7 min followed by decreasing the temperature to 4uC completed the PCR reaction. To verify the PCR products, 2 mL of DNA sample was loaded on 1% agarose for gel electrophoresis. PCR products were retrieved from the gel and double digested with XhoI and NotI. Meanwhile, double digestion of pmirGLO vectors with XhoI and NotI had been performed. The digested fragments from the vector and PCR products were purified and used to clone recombinant plasmids, including wt/Mut-CyclinD1-39-UTR, CyclinD1-39UTR-Mut1, and CyclinD1-39UTR-Mut2. To identify the recombinant plas- mids, the inserts were double digested with XhoI and NotI. Identification of miR-338-3p Target by Dual-luciferase Reporter Assay The reporter assay was performed according to Promega’s instructions. Briefly, growth medium was removed from cultured cells, fresh medium (100 mL/well) was added, and luciferase substrate (100 mL/well) was prepared. The plate was gently rocked at room temperature for 10 min, and firefly luciferase activity (hLuc) was then measured using a microplate luminometer (VeritasTM microplate fluorescence reader; YuanPingHao Bio, Beijing, China). Next, 100 mL of Stop & Glo reagent was dispensed, incubated for 10 min by gentle rocking, and Renilla luciferase activity (hRluc) was measured. The luciferase counts were then normalized to hLuc counts to obtain final reporter activity. Each sample was measured in triplicate. EdU Assay Cell proliferation was measured by 5-ethynyl-29-deoxyuridine (EdU) assay using an EdU assay kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Briey, LO2/HBx- d382 and LO2/HBx cells were cultured in triplicate at 56103 cells per well in 96-well plates and were transfected with 50 nM of miR-338-3p mimics, miR-338-3p inhibitor, or their respective control RNA for 48 h. The cells were then exposed to 50 mM of EdU for additional 4 h at 37uC. The cells were then fixed with 4% formaldehyde for 15 min at room temperature and treated with 0.5% Triton X-100 for 20 min at room temperature for permeabilization. After 36 washes with PBS, the cells were treated with 100 mL of 16 ApolloR reaction cocktail for 30 min. Subsequently, the DNA contents of each well of cells were stained with 100 mL of Hoechst 33342 (5 mg/mL) for 30 min and visualized under a uorescent microscope (Olympus, Japan). Cell Cycle Assay Cell cycle analysis was determined by flow cytometry (BD, UA). Briey, LO2/HBx-d382 and LO2/HBx cells at 16106 cells per well were cultured in 6-well plates and transfected with 50 nM of miR-338-3p mimics, miR-338-3p inhibitor, or their respective control RNA for 48 h. The cells were then harvested and fixed in 70% ice-cold ethanol for 24 h, followed by propidium iodide (PI) staining. The different cell cycle phases were analyzed using a FACS Calibur instrument. Statistical Analysis The data from soft agar colony formation and CyclinD1 expression in the engineered LO2 cells was performed with Fisher’s Least Significant Difference (LSD)-t test. The data from miR-338-3p expression in normal hepatocytes was performed with Fisher’s Least Significant Difference (LSD)-t test. All the other data was analyzed by unpaired two-tailed t-test. All data was expressed as means and standard deviation from at least 3 independent experiments. All p values were obtained with SPSS 16.0 software package, and a p,0.05 was considered statistically significant. Results HBx-d382 Enhances the Non-anchored Growing Ability of Transfected Hepatocytes and Promotes CyclinD1 Expression There is an established correlation between the presence of HBx deletion mutations in liver cells and hepatocarcinogenesis. To study whether and how the HBx deletion mutation affected the growth and proliferation of hepatocytes, we observed the isolated effect of the HBx-d382 HBx deletion mutant, found previously in our studies to be prevalent in HCC patient tumors, on hepatocyte cell proliferation as compared to the wild-type HBx gene. To do this, we established a non-tumorigenic human hepatocyte cell line, LO2, expressing the mutated HBx-d382 gene as well as a control LO2 cell line expressing the wild-type HBx. We first used RT- PCR to identify the HBx gene in the cDNA of the engineered LO2 cells. b-actin was used as a loading control (Fig. 1A). The data showed that the HBx gene had been successfully introduced into the host genome in LO2 cells. Western blotting showed that HBx protein expression could be detected in LO2 cells (Fig. 1B). These data suggest that stably HBx-transfected LO2 cells were successfully established. We used these engineered cells to test whether HBx-d382 had an effect on cell proliferation. Soft agar colony formation analysis revealed that the rate of colony formation in LO2/HBx-d382 and LO2/HBx groups was significantly higher than that of the control LO2 group and LO2/pcDNA3.0 groups after culturing for 2 weeks (p,0.01); there was no significant difference in colony formation between the control LO2 and LO2/pcDNA3.0 groups (Fig. 1C). Within the LO2/HBx-d382 and LO2/HBx groups, the former was higher than the latter (p,0.01). This result indicated that HBx and HBx-d382 led to cell cycle dysregulation, with the HBx-d382 mutant leading to the most pronounced proliferation PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43204