Ethics statement
The study protocol was approved by the ethics committee of The Second Affiliated Hospital of Nanchang University, and all HCC patients provided written informed consents regarding the use of clinical specimens for the study.
Sample collection and cell culture
Sixty-eight HCC tissue samples were collected from patients who underwent hepatectomy as treatment of HCC at The Second Affiliated Hospital of Nanchang University. Information pertaining to the clinicopathological parameters was also available. Liver cancer cell lines (HepG2, SMMC-7721 and Hep3B) were purchased from American Type Culture Collection (ATCC, USA) and the normal human hepatic cell line (LO2) was preserved in our laboratory and maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA), 100 U/ml of penicillin (Gibco, USA), and 100 μg/ml of streptomycin (Gibco, USA) at 5% CO2 and 37 °C. The medium was changed every 2 days, and cells were passaged at 70–80% confluence.
Cell transfection
The XPD overexpression plasmid, vector, miR-29a-3p inhibitor, inhibitor negative control (NC), miR-29a-3p mimic, mimic NC, Mdm2 overexpression plasmid or PDGF-B overexpression plasmid transfected into SMMC7721 cell. XPD siRNA, scramble, miR-29a-3p mimic or mimic NC, miR-29a-3p inhibitor or inhibitor NC, Mdm2 siRNA or PDGF-B siRNA were synthesized by GenePharma (Shanghai, China) and transfected into Hep3B cell. All cell transfections were introduced by Lipofectamine 2000 (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions. For each cell transfection, three replicates were performed.
Western blotting
Total proteins were extracted from Hep3B or SMMC-7721 cells using RIPA lysis buffer (Beyotime, China) and detected quantified with the BCA kit (Beyotime Biotechnology). Equal volume of protein were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking in PBS with 5% skim milk for 1 h at room temperature, the membrane was incubated overnight at 4 °C with corresponding primary antibodies including XPD (1:1000; Abcam, Cambridge, UK), Mdm2 (1:100, Calbiochem, Bad Soden, Germany), P53 (1:400, Bioworld Technology Inc., Massachusetts, USA) and PDGF-B (1:1000; Abcam, Cambridge, UK), furthermore, it was incubated for 2 h with horseradish peroxidase (HRP) conjugated secondary antibodies at room temperature and the ECL kit was used to detect immunoreactive bands according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA).
QRT-PCR
Total RNA was extracted from the transfected cells and frozen tissues using TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol. Reverse transcription was carried out using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). cDNAs were subjected to real-time PCR with use of Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocol. The results were calculated with the 2−△△Ct method.
Cell proliferation assay
The effect of XPD on SMMC7721 and Hep3B cell proliferation was measured by MTT assay. The cells were seeded in a 96-well plate at a density of 5000 monolayer cells per well. After 24 h, the cells were incubated with XPD for 24 h. Subsequently, the cells were washed with PBS and incubated with 20 µl MTT solution (5 g/l) for 4 h. After that, 150 µl DMSO (Shanghai Pharmaceutical Group, Shanghai, China) was added to each well to dissolve the crystals and then the plates were oscillated for 10 min in the dark. Finally, the optical density (OD) was measured at 490 nm using multifunctional fluorescence microplate reader. This experiment was performed in triplicate.
Cell migration assay
Cell migration was assessed by Transwell assays. Cells were suspended in 100 μl serum-free medium and were plated in the upper chamber of each insert (Corning, USA) with a Matrigel-coated membrane (BD Bioscience, San Jose, USA). The lower chambers of the inserts were filled with DMEM medium with 10% FBS. After 24 h of incubation, cells that migrated to the lower surface of the insert were fixed, stained with 20% methanol and 0.2% crystal violet, and counted under a light microscope (Olympus, Tokyo, Japan).
Luciferase reporter assay
Cells (5 × 104 cells/well) were cultured in a 24-well plate and co-transfected with wild type (Mdm2-WT, PDGF-B-WT) or mutant (Mdm2-Mut, PDGF-B-Mut), miR-29a-3p mimic and mimic NC using Lipofectamine 2000 (Invitrogen) for 48 h. Firefly activity was normalized to luciferase reporter plasmid (pRL-CMV). Renilla activity as control of transfection efficiency. The luciferase activities were measured by the dual-luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer’s instructions.
Animal experiments
All animal experiments were approved by the Ethical Committee on Animal Experiments at the The Second Affiliated Hospital of Nanchang University. For tumor growth assays, SMMC7721 cells treated with lentiviral vector of XPD overexpression, miR-29a-3p antagomiR, XPD overexpression + miR-29a-3p antagomiR or vehicle were subcutaneously injected into the right scapulas of nude mice (5-week-old BALB/c-nude, 8 per group, 2.0 × 106 cells for each mouse). The mice were observed over 34 days for tumor formation. The tumor volume was monitored every 3 days and calculated using the formula: V = 0.5 × length × width2.
Statistical analysis
All date were analyzed with SPSS 16.0. Data were presented as mean ± standard deviation (SD). Student’s t test was used to analyze differences between two groups. One-way ANOVA analysis was used to determine the multi-sample analysis. Differences at P < 0.05 were considered to be statistically significant.