Cell culture and transfection
The MDV-transformed chicken lymphoblastoid cell line MDCC-MSB1 was purchased from Shanghai Kindu Biotechnology Co. Ltd. The cells were cultured at 37°C under 5% CO2 in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal calf serum (Gibco, USA), 10% tryptose phosphate broth (Sigma, USA) and 1% penicillin-streptomycin solution (HyClone, USA). The gga-miR-155 mimic, gga-miR-155 inhibitor and their respective negative controls were synthesized by RiboBio Corporation (China). The constructs were transfected using FuGENE® HD (Promega, USA) according to the manufacturer’s instructions. Briefly, the cells were seeded in 6-well plates at the density of 3×105 per well, and transfected with 50nM gga-miR-155 mimic or 200nM gga-miR-155 inhibitor and their respective controls (NC). The transfection efficiency was validated after 48h by RT-qPCR.
Stem-loop quantitative real- time PCR (qRT- PCR)
Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol, and quantified using the NanoDrop ND-2000 Spectrophotometer (Thermo, USA). Reverse transcription was performed using the miRNA-specific stem-loop reverse-transcription primer (Sangon, Shanghai, China) using 1μg total RNA. Real time qPCR was performed using miScript SYBR Green PCR kit (Qiagen,USA) in the ABI 7900 PCR Detection System (Applied Biosysterm, USA). The cycling parameters were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 30 s and 60°C for 1 min. The relative target gene expression (2−ΔΔCt) was normalized to that of U6 endogenous small nuclear RNA. The primer sequences are shown in Table 1.
Target genes prediction and Luciferase reporter assay
The target genes of gga-miR-155 were predicted using the online tools TargetScan (http://www.targetscan.org) and miRDB (http://mirdb.org/miRDB/). RORA 3ʹ-UTR sequences containing the putative gga-miR-155 binding sites were amplified and cloned into the pYr-MirTarget luciferase reporter vector. The primers used are listed in Table 2. HEK293T cells in the logarithmic growth phase were seeded into 12-well plates and cultured overnight, and co-transfected with the RORA 3ʹ-UTR reporter vector and gga-miR-155 mimics or gga-miR-155 inhibitor using LipofectamineTM 2000. Luciferase activity was measured 48h after co-transfection using the Double Luciferase Reporter Gene Assay kit (Promega) according to the manufacturer’s instructions. The ratio of renilla and firefly luciferase intensities was calculated. The assay was performed thrice.
Total proteins were extracted from MSB1 cells 48h post-transfection using radio immunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors. The concentration of proteins was determined by the BCA assay (BCA Protein Assay Kit, Beyotime, Shanghai, China), and 20µg protein per sample was denatured in loading buffer by boiling for 3~5 min, and separated by 10% SDS-PAGE. The resulting bands were electro-transferred to polyvinylidene difluoride (PVDF) membrane at 100 mA over 1.5h. After blocking with 4% BSA for 1h, the membranes were incubated overnight with primary antibodies against RORA (1:1000, Abcam, ab60134) and β-actin (1:1,000, Abcam, ab8226), followed by HRP-conjugated anti-rabbit IgG (1:1000) and anti-mouse IgG (1:1000) (Bayotime) respectively. The positive proteins bands were detected using a chemiluminescence system (Bio-Rad Clarity Western ECL; Bio-Rad Laboratories Inc.), and the grayscale values were quantified using ImageJ. The density of the RORA bands was standardized to that of β-actin.
Cell proliferation assay
Suitably transfected MSB1 cells were harvested after 6h, and seeded into 96-well plates at the density of 4000 cells/well. Twenty microliters of the Cell Counting Kit 8 reagent (CCK 8, Biosharp Biotech, China) was added to each well after 6, 24, 48, 72 and 96 h of culture, and the optical density (OD) was measured at 450 nm using an ELISA reader (Thermo, USA) following a 4 h incubation.
Cell cycle assay
The cell cycle profile was analyzed using the Cell Cycle Detection Kit (KeyGen, China) according to the manufacturer's instructions. Briefly, the cells harvested 48 h after transfection were washed twice with cold PBS, and re-suspended in 100µl PBS. After fixing with 70% ice-cold ethanol for 4 h at 4°C, the cells were rinsed twice with cold PBS, incubated with 100μl RNase (50 μg/mL; Sigma, USA) for 30 min at 37°C, and finally stained with 400μl PI (50 μg/ml) in the dark at 4°C for 30 min. The stained cells were analyzed by flow cytometry (BD bioscience, USA). Each sample was tested in triplicates.
Cell migration and invasion assay
The in vitro migration and invasion of MSB1 cells were analyzed 48h after transfection using the transwell method. For the migration assay, RPMI 1640 medium containing 10% FBS was dispensed into the lower chambers of transwell inserts (8μm pore size; Corning 3422, USA) placed in a 24-well plate. After 1 h, 200 µL of the cell suspension (6×105 cell/ml) in serum free RMPI 1640 was seeded in the upper chambers of each insert. After incubating the cells for 24 h at 37°C under 5% CO2, the transwells were removed, and 60 µL CCK-8 reagent (Biosharp Biotech, China) was added to each well. The viability of the migrated cells was assessed as already described. The invasiveness of MSB1 was assayed as above, except that the seeding density of the cells was 1 × 105/well, and the upper chambers were pre-coated with Matrigel (BD Bioscience, USA). Each sample was tested in triplicates.
Apoptosis in the MSB1 cells was evaluated by AnnexinV-APC/7-AAD staining and flow cytometry (CytoFLEX; Beckman Coulter Inc., USA). Transfected cells were harvested after 48 h, washed twice with cold PBS, and stained using the AnnexinV-APC/7-AAD Cell Apoptosis Detection kit (NanJing KeyGen Biotech Co.,Ltd, China) according to the manufacturer’s instructions. The cells were resuspended in 500μl binding buffer,and incubated with 5 µL each of AnnexinV-APC and 7-AAD for 15 min in the dark at room temperature. The stained samples were analyzed by flow cytometry, and the percentage of apoptotic cells was calculated.
SPSS 19.0 and GraphPad Prism (Version 6.0) were used for data analysis. Data were expressed as mean ± SD. Two groups were compared using Student’s t-test, and multiple groups with the one-way ANOVA and LSD tests. P values < 0.05 were considered statistically significant.