Cell lines and tissue specimens
Human embryonic kidney cells (HEK293T),immortalized liver cells (L02) and human HCC cell lines including HepG2, MHCCLM3, MHCC97H, MHCC97L, PLC, HuH7 and Bel-7402 were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. Paired HCC and adjacent non-tumor liver tissues were collected from patients undergoing liver transplantation (LT) or partial hepatectomy at The First Affiliated Hospital, School of Medicine, Zhejiang University (Hangzhou, P.R. China). Written informed consent was obtained from each patient. A total of 125 patients had a clear histologic diagnosis of HCC with complete clinicopathological data, and all patients were closely followed up for survival analysis. None of the patients received radiotherapy or chemotherapy before surgery. All patients received the same anti-cancer treatment after operation. All sample data were obtained from the clinical and pathologic records and are summarized in Additional file
1: Table S1.
miR-503 mimic, miR-503 inhibitor and the respective control RNA (referred to as NC) were used for the transient gain- and loss-of-function study. The small interfering RNA (siRNA) targeting human cyclin D3 (GenBank Access. No. NM_001136017) and E2F3 (NM_001949) transcripts were designated siCCND3 and siE2F3 respectively. The NC for miR-503 mimic, miR-503 inhibitor and siRNA was non-homologous to any human genome sequence. For the in vivo and in vitro tumorigenicity assay, all nucleotides with 2′-O-methyl modification were used. All the RNA oligoribonucleotides (Additional file
2: Table S2) were purchased from Genepharma (Shanghai, China). All oligoribonucleotides used in this study are shown in Additional file
2: Table S2.
RNA extraction and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
Total RNA from cell lines and clinical samples was isolated using the mirVana miRNA isolation kit (Ambion). Quantitative reverse transcriptase polymerase chain (qRT-PCR) was performed to evaluate the expression level of miR-503 in various cell lines and clinical samples and the expression of cyclin D3 and E2F3 in transfected cells. RNA was reverse transcribed using One Step PrimeScript miRNA cDNA Synthesis Kit (TaKaRa, Japan). The cDNA was then quantified by real-time RT-PCR using SYBR Premix Ex Taq (TaKaRa, Japan). All PCR reactions were performed using the ABI7500 system (Applied Biosystems, CA, USA). RNU6B or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control, and miR-503 expression values were normalized to RNU6B. All primers used are listed in Additional file
2: Table S2.
Western blotting was used to detect the expression of target genes at the protein level. Protein was extracted from transfected MHCCLM3 cells using modified RIPA buffer in the presence of proteinase inhibitor cocktail. Equivalent quantities (30–50 μg) of protein were separated in 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk and then incubated overnight at 4°C with the appropriate primary antibody at the dilutions specified by the manufacturer. The membranes were then washed three times in 10-ml TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at 1:2000 dilution for 1 h. Bound secondary antibody was detected using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology Inc., Rockford, IL, USA). Primary antibodies were as follows: anti-E2F3 (Abcam), anti-cyclinD3, anti-Rb, anti-phospho-Ser780-Rb, anti-CDK4, anti-CDK6, anti-cyclin A, anti-cdc2, anti-p15, anti-p16 (Cell Signaling Technology), anti-phospho-Ser811-Rb, and anti-β-actin (Epitomics).
The transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In brief, MHCCLM3, HepG2, Bel-7402 or HEK293T cells were transfected with DNA, miRNA mimic, miRNA inhibitor, siRNA or respective NC. All RNA transfections were performed at a final concentration of 50 nM. Cells were collected for assay 48 h after transfection. The transfection efficiency of the miR-503 mimic and inhibitor was confirmed by detection of miR-503 expression using qRT-PCR.
Cell proliferation assay
MHCCLM3, HepG2 and Bel-7402 cells seeded at a density of 4,000 per well into 96-well plates were transfected with miR-503 mimic, miR-503 inhibitor or respective NC. After incubating the cells for the specified time (1, 2, 3 or 4 days), a cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8) (Dojindo) according to the manufacturer’s instructions. The solution absorbance was measured spectrophotometrically at 450 nm with MRX II absorbance reader (Dynex Technologies, Chantilly, VA, USA). The experiments were performed in triplicate.
Analysis of clonogenicity in vitro and tumorigenicity in nude mice
Aliquots of viable MHCCLM3, HepG2 and Bel-7402 cells (1,000 per well) transfected with miR-503 mimic, miR-503 inhibitor or respective NC were placed in six-well plates 24 h after transfection and maintained in complete medium for 2 weeks. Colony formation was evaluated by staining the cells with 0.1% crystal violet. The rate of colony formation was calculated using the following equation: colony formation rate = (number of colonies/number of seeded cells) × 100%. The experiments were performed in triplicate.
Animal study was performed according to institutional ethical guidelines. Male BALB/c-nude mice aged 4 weeks were used for human tumor xenograft model (supplied by the Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China). Viable miR-503- or NC-transfected MHCCLM3 cells (5×106) were suspended in 100 μl PBS and then injected subcutaneously into the posterior flank of nude mice, respectively. Tumor growth was examined every week for 5 weeks. Tumor volume (V) was monitored by measuring the length (L) and width (W) with calipers and calculated with the formula (L × W2) × 0.5.
Cell cycle analysis by flow cytometry
Forty-eight hours after transfection, 1×105 MHCCLM3, HepG2 and Bel-7402 cells transfected with miR-503 mimic, miR-503 inhibitor or respective NC were harvested, washed with PBS and fixed in 70% ethanol at 4°C overnight. Staining for DNA content was performed using a DNA Prep Stain (Beckman Coulter, Fullerton, CA, USA). Populations in G0/G1, S and G2/M phases were measured by BD LSRII Flow Cytometry System with FACSDiva software (BD Bioscience, Franklin Lakes, USA). Data were analyzed using the ModFit LT Software. The experiments were performed in triplicate.
Vector construction and luciferase reporter assay
The 3′ UTR of cyclin D3 or E2F3 was amplified by PCR. The amplified product was subcloned and ligated into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). The recombinant reporter vector was identified by sequencing and termed the wild-type (Wt). To create the miR-503 binding site mutants, the binding region of the seed sequence (5′ GCTGCT 3′) was mutated to the sequence 5′ CGACGA 3′ (mutated nucleotides are in bold and underlined), using the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s protocol. The recombinant vector was confirmed by sequencing and termed the mutant type (Mut). 293T cells plated in a 24-well plate were co-transfected with 50 nM of either miR-503 mimic or NC and 100 ng of pmirGLO vector comprising Wt or Mut 3′ UTR of cyclin D3 and E2F3 by Lipofectamine 2000. Forty-eight hours after transfection, the relative luciferase activity was measured by Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. All transfection experiments were performed in triplicate.
Data are shown as the means ± SEM of at least three independent experiments. Differences between groups were analyzed using Student’s t-test, the χ2 test and the log-rank test when two groups were compared. Overall survival rates were calculated actuarially according to the Kaplan–Meier method. Variables with a value of p < 0.05 in univariate analysis were used in a subsequent multivariate analysis based on the Cox proportional hazards model. All tests performed were two-sided. A value of p < 0.05 was considered to indicate statistical significance.