Cells, viruses, plasmids and reagents
Human lung diploid fibroblastic KMB17 cells (Institute of Medical Biology, CAMS, Kunming, China) were grown in Minimum Essential Medium (MEM) supplemented with 10% bovine serum (Minhai Biotech, Beijing, China) at 37°C in a humid environment containing 5% CO2. HEK293T cells were purchased from Thermo Scientific (Cat no. HCL4517) and grown in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose formulation, Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Gibco Life Technologies, Grand Island, NY, USA) at 37°C in a humid environment containing 5% CO2. Human hepatitis A virus strain H2 (lg107.6 TCID50/ml), an attenuated vaccine strain (Institute of Medical Biology, Kunming, CAMS) was prepared in KMB17 cells .
The eukaryotic expression plasmid pcDNA6.2-GW/EmGFP-hav-pre-miRNA-50 obtained by inserting the entire viral pre-hav-miR-N1-3p sequence into pcDNA6.2-GW/EmGFP-miR vector (Invitrogen, Carlsbad, CA, USA) was constructed via Gateway cloning using BLOCK-iT™ Pol II miR RNAi Expression Vector Kits (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. The pmirGLO-hav-miRNA target expression plasmids wild-type (sensor) and mutant type (sensor-mut) were generated by inserting two tandem repeats of the antisense sequence of viral miRNA or cellular miR-154 (or mutated version) into 3' UTR of the luciferase gene of the pmirGLO vector (Promega, Madison, WI, USA), according to the manufacturer’s protocol. All constructs generated were confirmed by sequencing using universal primers (BGI, Guangzhou, China).
Mouse monoclonal anti-Dicer1 antibody 5D12.2 (1:5000 dilution; mouse monoclonal; Millipore Corporation, Billerica, MA, USA), rabbit anti-actin polyclonal antibody (20536-1-Ap; 1:2000 dilution; rabbit polyclonal; Proteintech Group, lnc. Chicago, IL, USA), and appropriate HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (1:10,000 dilution; Proteintech Group, lnc. Chicago, IL, USA) were used for immunoblotting.
Bioinformatics prediction of the miRNAs
A flowchart describing the computational prediction of putative miRNAs is shown in Figure 1. Briefly, the viral antigenome was scanned for miRNA precursors (pre-miRNA) stem-loop structures using VMir, a computational analyzer program [16, 35, 36] for prediction of putative pre-miRNAs. Six complete antigenome sequences of different cell-adapted passaged HAV strain H2 (GenBank accession no. EF406358.1, EF406359.1, EF406360.1, EF406361.1, EF406362.1, EF406363.1) were used . VMir predictions were carried out using default parameters. The putative pre-miRNAs that fulfilled filter parameters with VMir score ≥ 150 and window counts ≥ 35 were selected for further assessment. Subsequently, mature miRNA sequences from pre-miNRA stem-loops were predicted (Additional file 1). In order to extend the prediction coverage of the mature miRNAs, we performed two strategies: the MatureBayes tool  (http://mirna.imbb.forth.gr/MatureBayes.html) and Bayes-SVM-MiRNA web server v1.0 (http://wotan.wistar.upenn.edu/BayesSVMmiRNAfind/). Default conditions were followed for the MatureBayes tool. Folding energy was set at -15 kcal/mol when using Bayes-SVM-MiRNA web server v1.0; other filter parameters were set to default values.
Stem-loop RT-PCR analysis and PCR-based directed cloning of the miRNAs
KMB17 cells were infected at a multiplicity of infection (MOI) of 1.0 50% tissue culture infective doses (TCID50) /cell of HAV strain H2. Mock-infected cells was used as negative control. At 24 hours post-infection, total RNA was extracted with the Trizol reagent (Invitrogen China Ltd., Shanghai, China) according to manufacturer’s protocol. A highly sensitive and specific stem-loop RT-PCR was used to detect the expression of the candidate miRNAs as described previously [39, 57], with minor modifications. Briefly, first strand cDNA of miRNAs were synthesized using stem-loop RT primers (Additional file 3) and Reverse Transcription System (Cat. no. A5001; Promega, Madison, WI, USA), following the manufacturer’s instructions. Then, miRNA cDNAs were amplified by PCR in a mixture including rTaq DNA polymerase (TaKaRa, Dalian, China). The reaction mixture was subjected to 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 30 sec. A cellular miR-154 was used as a positive control for miRNA size when products were separated by agarose gel electrophoresis. In addition, to determine the specificity of qPCR products, agarose gel electrophoresis analysis and T-A cloning strategy were conducted. The PCR products were analyzed on 4% agarose gel and purified PCR products were subcloned into the pGEM-T vector (Promega, Madison, WI, USA) and sequenced to verify the exact miRNA sequences. KMB17 cells were infected at MOI of 0.5 TCID50/cell of HAV. Cells infected at various time points 0, 4, 8, 12, 24, 48, 72, 96, 120 hours were analyzed for time course expression of miRNA. Stem-loop qRT-PCR was performed to quantify miRNA levels using Reverse Trancription System (Cat. no. A5001; Promega, Madison, WI, USA) and GoTaq qPCR kit (Cat. no. A6001; Promega, Madison, WI, USA). Cellular U6 snRNA was as an endogenous control. Relative expression levels were calculated using the 2-ΔΔCt method for infected versus uninfected cells .
RNA interference (RNAi) of the Dicer gene
The siRNA duplexes against the Dicer gene were synthesized according to sequences reported by Moore, et al.
 and Bennasser, et al.
 (Additional file 4). In order to avoid off-target effects, siRNAs were designed for multiple targets of the target gene. All siRNAs used for knockdown of the target gene and scrambled siRNA (a negative control) were chemically synthesized with 2' OME modification by GenePharma (Shanghai, China). siRNAs were dissolved in 0.1% diethylpyrocarbonate (DEPC) treated water to a final concentration of 20 μM and stored at -80°C. KMB17 cells were seeded in 6-well plates one day before transfection. The next day, when the cells reached approximately 50-70% confluence, 100 pmol siRNA against Dicer mRNA or non-specific negative control siRNA were transfected into KMB17 or HEK293T cells using Lipofectamine 2000 (Invitrogen China Ltd., Shanghai, China), according to the manufacturer’s protocol. Seventy-two hours after transfection, siRNAs transfected cells were lysed with RIPA buffer (Pierce, Rockford, IL, USA) containing the protease inhibitor PMSF (Solarbio, Beijing, China). Total cell protein extracts were collected for Western blot analysis.
Real-time quantitative RT-PCR
The expression levels of viral miRNA and the Dicer gene were analyzed by real-time quantitative RT-PCR (qRT-PCR) with specific primers (Additional files 3 and 5) using the Reverse Trancription System (Cat. no. A5001; Promega, Madison, WI, USA) and GoTaq qPCR kit (Cat. no. A6001; Promega, Madison, WI, USA) according to the manufacturer’s instructions. For viral miRNA, cellular U6 snRNA gene was determined by qRT-PCR in parallel as an internal standard control. Total RNA was analyzed on a CFX96™ Real-Time PCR system (Bio-Rad, CA, USA) using the following program: 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 56°C for 15 sec, and extension at 72°C for 30 sec. Relative miRNA abundance was normalized against cellular U6 snRNA content and assessed by the 2-ΔΔCt method . For the Dicer gene, cellular Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was determined by qRT-PCR in parallel as an endogenous control. Total RNA was analyzed on a CFX96™ Real-Time PCR system (Bio-Rad, CA, USA) under standard conditions using the following program: 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 42°C for 15 sec, and extension at 72°C for 30 sec. Relative abundance of target gene mRNA was normalized to cellular GAPDH mRNA content and assessed by the 2-ΔΔCt method . Melting curve analysis was also carried out to determine qPCR product specificity. The Ct (cycle threshold) values were determined using default threshold settings. All qRT-PCR assays were performed with three biological replicates and three technical replicates.
Western blot analysis
Total protein in cell extracts was quantitated by the BCA protein assay Kit (Pierce, Rockford, IL, USA) according to manufacturer’s instructions. Thirty micrograms total protein were resolved on 8% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% non-fat milk at room temperature for 2 hours. Membranes were probed with specific primary antibody overnight at 4°C, followed by incubation with appropriate (anti-mouse or anti-rabbit) HRP-conjugated secondary antibody at room temperature for 1 hour. Protein signals were visualized by ECL chemiluminescence using Immobilon Western HRP Substrate (Millipore Corporation, Billerica, MA, USA) according to the manufacturer’s protocol.
Luciferase reporter assay
A dual luciferase reporter assay using pmirGLO-hav-miR-N1-3p sensor and pmirGLO-hav-miR-N1-3p-sensor-mut was performed in HEK293T and KMB17 cells. Briefly, HEK293T cells were seeded at approximately 1 × 106 cells per well in a 24-well plate one day before transfection. The next day, 200 ng pmirGLO-hav-miR-N1-3p-sensor or pmirGLO-hav-miR-N1-3p-sensor-mut plasmid were co-transfected with 20 pmol chemically synthesized miRNA mimics (GenePharma, shanghai, China) (Additional file 6) into HEK293T cells with Lipofectamine 2000 (Invitrogen). A cellular miR-154 served as a positive control, and mutated pmirGLO-hav-miR-N1-3p-sensor-mut was used as a negative control. Additionally, KMB17 cells prior to infection with HAV (MOI = 10.0 TCID50/cell) were seeded at approximately 1 × 106 cells per well in a 24-well plate one day before transfection. The next day, 200 ng wild type and mutated reporter sensor plasmids were transfected into HAV-infected and mock-infected KMB17 cells. The firefly and Renilla luciferase activities were evaluated simultaneously 48 hours post-transfection using the Dual-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Relative luciferase activity was expressed as the ratio of firefly to Renilla luciferase activity. The transfections were performed independently, in triplicate.
Values from three independent experiments were analyzed by two-tailed Student’s t-test. P < 0.05 was considered statistically significant and P < 0.01 highly statistically significant.