Cell culture and siRNA transfection
HepG2 cells were obtained from the School of Medical Sciences, Zhengzhou University, and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone, Logan, UT, USA) with 10% foetal bovine serum (FBS) (HyClone), 100 µg/ml penicillin, and 0.25 µg/ml streptomycin at 37 °C and 5% CO2.
siRNA targeting the cyclin G1 gene and the negative control siRNA were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), and the cyclin G1 siRNA sequence was as follows:
anti-sense: 5′-UAAACAUUUGGAUACAAGCUCUUGCCA − 3′. The negative control siRNA sequence was as follows:
sense: 5′-CUUCCUCUCUUUCUCUCC CUUGUGA-3’,
antisense: 5’-UCACAAGGGAGAGAAAGAGAGGAAGGA-3’. According to the manufacturer's protocol, the siRNA sequences were transfected with the Lipofectamine 2000 transfection reagent.
Total RNA was extracted using TRIzol (GenePharma, Co., Ltd., Shanghai, China) and reverse transcribed to cDNA. qPCR was performed in a 7300 Sequence Detection System (Applied Biosystems, Foster City, USA) using a SYBR® Green PCR kit (QIAGEN, Hilden, Germany). The primers were purchased from Shanghai GenePharma Co., Ltd. as follows:
cyclin G1: forward 5′-AGCTGCAGTCTCTGTCAAG-3′,
reverse 5′-ATGTCTCTGTGTCAAAGCCA − 3′;
and β-actin: forward 5′-GAAATCGTGCGT GACATTA-3′,
reverse 5′-ACTCATCGTACTCCTGCTTG-3′. The relative expression level of cyclin G1 was calculated using comparative cycle threshold (Ct) methods, and β-actin was used as an internal control. The value of cells transfected with negative control siRNA was set to 100% in each run.
Cell proliferation assay
Cells were seeded into 96-well plates at 1.5 × 103 cells and 2 × 103 cells per well and incubated overnight. The cells were cultured for 5 days at 37 °C. 3-(4,5-Dimethylthiazol − 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out at different time points: 24 h, 48 h, 72 h, 96 h and 120 h. Then, 20 µl MTT solution (5 mg/ml) was added to each well and incubated for an additional 4 h at 37 °C. Then, the MTT solution was aspirated, and 100 µl DMSO was added to dissolve the formazan crystals. The number of cells was counted using a microplate reader at a wavelength of 490 nm.
Colony formation assays
Cells were digested, and single-cell suspensions were prepared; then, cells were plated in 6-well plates at appropriate dilutions (200, 200, 400, 400, and 800 cells/well for the 0 Gy, 2 Gy, 4 Gy, 6 Gy and 8 Gy groups, respectively) and allowed to attach overnight. The cells were irradiated with different dosages using an ONCOR linear accelerator (Siemens, MUC, Germany) at a dose rate of 300 cGy/min, cultured for another 14 days, fixed with methanol, and stained with 0.05% crystal violet (Sigma-Aldrich Chemical Company, St Louis MO, USA). The number of colonies consisting of 50 or more cells was counted, and the surviving fraction was calculated. The dose-survival curves were plotted with a single-hit multitarget model using GraphPad 5 (GraphPad Software Inc., San Diego, CA, USA), and the values of D0, Dq and SERDq were calculated.
Xenograft transplantation and irradiation
To develop xenograft tumours, HepG2 cells, HepG2-cyclin G1-siRNA negative control cells and Hep-cyclin G1-siRNA cells (1.0 × 105 cells) were subcutaneously implanted into BALB/c nude mice (n = 5). When xenograft tumours grew to a mean volume of approximately 50 mm3, tumours were treated with 20 Gy radiation in 5 fractions (4 Gy per fraction, once every 2 days). Each animal was earmarked and followed individually throughout the experiment. Tumour volume (mm3) was calculated using the following formula: V(mm3) = length (mm) × width (mm)2/2. The body weights of the mice were also recorded every 2 days during the experiment. All tumours were observed for 21 days, and then the mice were sacrificed, and the weights of the tumours were weighted.
Western blot analysis
Membrane protein was extracted 48 h after irradiation, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-Page) and blotted onto a polyvinylidene difluoride membrane (Bio-Rad, CA, USA). The membranes were blocked with 5% milk for 2 h and incubated overnight with antibodies specific to cyclin G1 (1:1000), Bcl-2 and Bax (Cell Signaling Technology, MA, USA) (1:1000) or GAPDH (Abcam, MA, UK) (1:5000) at 4 °C. Proteins were visualized by using the electro-chemi-luminescene(ECL) procedure (Bio-Rad, CA, USA). The results were analysed using Quantity One software (Bio-Rad, CA, USA).
Data are expressed as the mean ± standard deviation (SD) and were analysed using the Statistical Program for Social Sciences (SPSS) 10.0 software (IBM, Armonk, NY, USA). Differences between groups were determined using analysis of variance (ANOVA) or Student’s t-test. A p-value less than 0.05 was considered statistically significant.