miRNA expression is associated with a variety of tumors, and that these miRNAs may play a tumor suppressor or oncogenic role [11–12]. Important to our present study, miRNAs have been reported to be closely related to the incidence and development of liver cancer [13–15]. However, only a few studies have evaluated the specific roles of either the HBx deletion mutants or miRNAs, or their possible interaction, in the incidence and development of HBV-related liver cancer. Our previous studies found that liver cancer tissues from chronically HBV-infected human patients contained an elevated incidence of a particular HBx deletion mutation, HBx-d382 (named because of a deletion mutation between nt 382–400) [16]. We speculated that this relationship was related to the higher incidence of HBx mutations that occur following HBV DNA integration, which often happens during chronic HBV infection that also correlates with the development of hepatocarcinoma. Upon further investigation by miRNA microarray analysis, we found that stable expression of HBX or HBx-d382 in LO2 cells decreased miRNA expression, including miR-338-3p, as com- pared to control LO2/pcDNA cells; interestingly, the LO2/HBx- d382 cells exhibited this phenotype in a more pronounced way than LO2/HBx cells. Additional analysis by real-time PCR confirmed this result (unpublished data). Furthermore, the miRNA target prediction based on bioinformatic analysis showed that CyclinD1 is a likely miR-338-3p target. To better understand the influence of the down-regulated miR-338-3p expression in cells expressing the HBx-d382 mutant and to determine whether there are any changes in miR-338-3p-mediated CyclinD1 expression, we used the stably transfected LO2/HBx and LO2/HBx-d382 (also used in our previously published experiments) as our experimental model. The results of our study suggest that the miR-338-3p-mediated CyclinD1 expression changes downstream of HBx mutant gene function may help to understand the mechanisms underlying the HBx-d382 mutant-induced hepato- carcinogenesis from a new perspective. Materials and Methods Cells Culture and Establishment of the Stably Transfected Cell Lines All cells were cultured in RPMI-1640 medium (GIBCO, USA) containing 10% fetal bovine serum (FBS) (GIBCO, USA) and 100 units/mL penicillin plus 100 mg/mL streptomycin at 37uC with 5% CO2. The control plasmid pcDNA3.0, recombinant plasmid pcDNA3.0/HBx-d382 (a mutant of the HBx gene with deletion from 382–400 bp), and pcDNA/HBx were previously established in our lab [16]. The HBx genetic fragments used here originate from the liver cell line HepG2.215. We established stably engineered LO2 cells transfected with the wild type HBx and HBx-d382 mutant plasmids; the hepatocyte cell line LO2 (obtained from Chinese Academy of Science, Cell Biology of Shanghai Institute) was transfected with LipofectamineTM2000 (Invitrogen, California, USA) according to the manufacturer’s instructions and was selected with G418 (Geneticin, GIBCO, USA). Empty pcDNA3.0 vector plasmid was used as a control. The stable transfection of pcDNA3.0/HBx-d382 (termed LO2/ HBx-d382), pcDNA/HBx (termed LO2/HBx), or the empty vector (termed LO2/pcDNA3.0) was confirmed by RT-PCR for gene expression and western blotting for protein expression. Cell Transfection HBx-expressing cells were transfected with miRNA mimics or miRNA inhibitor (GeneCopoeia, USA) using Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM, according to MiR-338-3p on HBx Deletion Mutants (HBx-d382) manufacturer’s instructions. The normal recommended controls for the miRNA-mimics and inhibitor (GeneCopoeia, USA) were used in the experiments. Changes in gene expression were detected 48 hr after transfection. miR-338-3p miRNA target verification was performed in LO2/HBx cells, which were co- transfected with miR-338-3p mimics and the dual luciferase reporter plasmid. miRNA-NC miRNA was used as the control for miR-338-3p. Inhibition of HBx Expression Small interfering RNA (siRNA) sequences specifically targeting HBx were selected accordingly [17] and synthesised by Gene- Pharma (Shanghai, China). Approximately 50 nM HBx siRNA or control siRNA was transfected into LO2/HBx and LO2/HBx- d382 cells by Lipofectamine as described above. RT-PCR Analysis Total RNA from the engineered LO2 cells was extracted by Trizol (Invitrogen, USA). The PCR primers used for HBx detection were as follows: for HBx, 59-AAGGTAC- CATGGCTGCTAGGCTGTGCT-39 (forward) and 59- CTGGGCCCTTAGGCAGAGGTGAAAAAGTTG-39 (reverse), leading to a 462 bp amplified product; for the b-actin control, 59- CTCCATCCTGGCCTCGCTGT-39 (forward) and 59- GCTGTCACCTTCACCGTTCC-39 (fragment), leading to a 242 bp amplified product. Soft Agar Colony Formation Assay The assay was conducted according to previously published methods [18], with slight modifications. Briefly, 56103 transfected LO2 cells were first thoroughly mixed with 2 mL RPMI Medium 1640 containing 3 g/L agar and 10% FBS. This mixture was then added onto solid agar (RPMI Medium 1640 medium containing 5 g/L agar and 10% FBS) in a 6-well plate and incubated for 2 weeks. Finally, the derived clones from each group within a randomly selected area were selected and counted under a microscope at 506 magnification. Quantitative Real-time PCR (qRT-PCR) Analysis miR-338-3p expression in normal hepatocytes (LO2 and QSG7701 cells) and HBx-expressing cells after knocking down HBx expression was measured with SYBR qRT-PCR. CyclinD1 expression before and after miR-338-3p mimic or inhibitor introduction into HBx-expressing LO2 cells was measured with SYBR qRT-PCR. Total RNA was extracted with Trizol (Invitrogen) according to the manufacturer’s instructions. miR-388-3p cDNA was synthesized from 2 mg of total RNA with an All-in-oneTM miRNA First-Strand cDNA Synthesis (GeneCopoeia) Kit using the supplied poly-A primer. Real-time PCR was performed in a 20 mL reaction mix including 2 mL of 56 diluted reverse transcription product, 2 mL miRNA specific primer, 10 mL SYBR 26 All-in-one qPCR Mix, 0.4 mL 506 ROX Reference dye, and 3.6 mL double distilled water. The cycling conditions for amplification on the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) were 95uC for 10 min, followed by 40 cycles of 95uC for 10 sec, 60uC for 20 sec, and 72uC for 32 sec. The data were normalized against the U6 snRNA. CyclinD1 expression was analyzed with THUNDERBIRD SYBR qPCR Mix (ToYoBo, Japan). cDNA was synthesized with the RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, Canada) in a total volume of 20 mL. The primer sequences used were as follows: for CyclinD1, 59-AGGAACA- PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43204