The human cervical cell line C33A was obtained from the Shanghai Life Science Institute Cell Library (Shanghai, China). C33A and the acquired radioresistant cell line C33AR were cultured in Minimum Essential Medium (MEM) (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences), penicillin-streptomycin liquid (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 0.25 µg/ml amphotericin B (Amresco, Inc., Framingham, MA, USA) in a humidified atmosphere of 5% CO₂ at 37°C. The miRNA-320 agomir (a novel class of chemically engineered miRNA mimic) and negative control were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were transfected with 100 nM miRNA-320 agomir or negative control using Lipofectamine 2000 in Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A miRNA-320 inhibitor antiagomir (Guangzhou RiboBio Co., Ltd.) was designed to suppress the expression of miRNA-320. Small interference RNA (siRNA) was used to inhibit β-catenin expression. The siRNAs against β-catenin as well as the non-targeting control siRNAs were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells were transfected with siRNAs at a concentration of 25 nM using Lipofectamine 2000. The sequence of the β-catenin siRNA was 5′-GGACACAGCAGCAAUUUGUTT-3′.

Cells were cultured to 80% confluence and then subjected to irradiation. Irradiation parameters were set as follows: Quality, 6 MV/X-rays; dose rate, 2 Gy/min; field, 35×35 cm; and 2 Gy each time, using a linear accelerator. Cells were kept at room temperature for ≤30 min during irradiation. One set of flasks was not irradiated and treated as wild type. After 24 h of incubation, the medium in each flask was exchanged for fresh medium to remove detached cells. Cells resistant to radiation were cultured in the same medium, which was exchanged for fresh medium every 2 days thereafter. To generate stable radioresistant clones, all the clones after 60 Gy radiation were expanded for >6 months without radiation to confirm the radioresistant phenotype before studies were undertaken. Two clones from C33A cells were established. The parental cells of wild-type stable clones were generated under the same conditions without irradiation.

Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For the detection of miRNAs, RNA was reverse transcribed using a specific RT primer for U6 and miRNA-320 (Guangzhou RiboBio Co., Ltd.) according to the protocol of the manufacturer. qPCR was carried out with SYBR Green I Mix (Takara Biotechnology Co., Ltd., Dalian, China) in a 20-µl reaction volume (12.5 µl SYBR Green I Mix, 0.5 µl ROX reference dye II, 200 mM forward and reverse primer, 2 µl complementary DNA template and 8 µl double-distilled H₂O) on the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the following protocol: 95°C for 20 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 70°C for 10 sec. The primers for miR-212/320/132/15a/16 for RT-qPCR were designed and synthesized by Guangzhou RiboBio Co., Ltd. The sequences for the remaining primers were as follows: β-catenin forward, 5′-GAAACGGCTTTCAGTTGAGC-3′ and reverse, 5′-CTGGCCATATCCACCAGAGT-3′; c-MYC forward, 5′-GGGTAGTGGAAAACCAGCAGC-3′ and reverse, 5′-CCTCCTCGTCGCAGTAGAAATA-3′; cyclin D1 forward, 5′-GAGGAACAGAAGTGCGAGGAG-3′ and reverse, 5′-GGATGGAGTTGTCGGTGTAGAT-3′; and GAPDH forward, 5′-TGGAAGGACTCATGACCACA-3′ and reverse, 5′-TTCAGCTCAGGGATGACCTT-3′. ∆ quantification cycle (Cq) was calculated by subtracting the Cq of U6 from the Cq of miRNA-320. ∆∆Cq was then calculated by subtracting the ∆Cq of the control from the ∆Cq of the treatment group. The fold-change of miRNA expression was calculated by the equation 2^−∆∆Cq (10).

Cells were harvested and washed with cold PBS three times, fixed with 70% ethanol at 4°C for 24 h, washed again three times with cold PBS and then stained with 50 mg/ml propidium iodide for 0.5 h at 37°C (Beyotime Institute of Biotechnology, Haimen, China). DNA content was analyzed on the Cytomics FC 500 (Beckman Coulter, Inc., Brea, CA, USA).

Cells in an exponential growth phase were plated into a 6-well plate at 100, 200, 400, 800, 1,000 and 2,000 cells per well, and then irradiated with 0, 2, 4, 6, 8 and 10 Gy. Cells were then incubated at 37°C in 5% CO₂, 95% air for 9–12 days. When most cell clones had reached >50 cells, they were stained with 0.5% (w/v) crystal violet (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The number of clones in the radioresistant group was compared with that in the wild-type group. The survival curves were obtained and analyzed with GraphPad Prism 4 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). Three independent experiments were performed.

Cell proliferation was assessed using the WST-1 Cell Proliferation Reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. The C33AR cells were plated into a 96-well cell culture plate at 2,000 cells/well and then incubated at 37°C overnight to allow settling. Then, they were transfected with miRNA-320 agomir and negative control, and treated with 4 Gy X-ray radiation. Subsequently, 10 µl of WST-1 reagent was added in each well and incubated for 2 h at 37°C. Absorbance was subsequently determined at a wavelength of 450 nm (for measurements) and 650 nm (as reference) by a microplate reader (EnSpire; PerkinElmer, Inc., Waltham, MA, USA). Cell proliferation was calculated by subtracting the absorbance values of the samples from that of the medium alone (background level). The relative cell proliferation was normalized to that of the control group.

The β-catenin siRNA oligonucleotides with the sequence si-1 sense 5′-GCAGUUGUAAACUUGAUUATT-3′; si-2 sense 5′-CCCAAGCUUUAGUAAAUAUTT-3′; and si-3 sense 5′-GGACACAGCAGCAAUUUGUTT-3′, along with the corresponding antisense oligonucleotides, were synthesized by Shanghai GenePharma Co., Ltd. A negative control siRNA (Shanghai GenePharma Co., Ltd.) was used as a control siRNA. siRNA transfection was performed using Lipofectamine 2000 as indicated in the manufacturer's instructions. Briefly, subconfluent C33AR cells were plated in 6-well plates in regular growth medium. The next day, they were transfected with either 100 nM control siRNA or the β-catenin siRNAs for 6 h, followed by recovery in serum-containing medium. After 48 h of siRNAs transfection, the cells were harvested for protein isolation analysis.

Cells were washed with PBS and lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA and a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Cell lysates were quantified for protein content by the bicinchoninic acid (BCA) method using a BCA kit (Bio-Rad Laboratories, Inc.). Then, 40 µg of protein was resolved in 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 5% bovine serum albumin (Beyotime Institute of Biotechnology) in TBS with Tween-20 (TBST) for 2 h, and then probed with primary antibodies against β-catenin (ab22656; Abcam, Cambridge, MA, USA), cyclin D1 (ab134175; Abcam), c-MYC (ab32072; Abcam) and GAPDH (KM1002T; Sungene Biotech Co., Ltd., Tianjin, China) at a 1:1,000 dilution at 4°C overnight, washed extensively with TBST three times, and incubated with secondary antibodies conjugated with horseradish peroxidase (Pierce; Thermo Fisher Scientific, Inc.)at room temperature for 2 h at a 1:5,000 dilution. Immunoreactive protein was examined using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology).

The results of the quantitative data in this study are expressed as the mean ± standard deviation. The Student's t-test was used to evaluate the significant difference between two groups of data in all pertinent experiments with the statistical package SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 (using a two-tailed paired t-test) was considered to indicate a statistically significant difference between two groups of data.

To generate a radioresistant cell line, C33A cells in exponential growth phase were exposed to X-rays at a dose of 2 Gy and a dose rate of 2 Gy/min. An interval of 1 to 3 weeks between each ionizing radiation (IR) allowed the surviving cells to regenerate. The whole process of IR and culture lasted for ~1 year, and the surviving cell line was termed C33AR. To verify phenotypes, C33AR cells were irradiated and examined by the colony-formation assay. Compared with C33A, C33AR showed no change in foci formation when IR was absent, but gained more foci and higher survival fractions when exposed to IR (Fig. 1A), establishing C33AR as a stable radioresistant cell line. Next, flow cytometry was used to evaluate alterations in the cell cycle (Fig. 1B and C). After irradiation, C33AR cells in the G1 and S phases significantly increased. Conversely, the proportion of C33AR in the G2/M phase decreased.

The differential miRNA expression profile between C33AR cells and their parental C33A cells was determined using RT-qPCR (Fig. 2A). The expression of miRNA-320 in C33AR cells was significantly lower than that in C33A cells. Several differentially expressed miRNAs in this profile were previously reported to have a role in tumorigenesis, including the development of radioresistance (17,18). These results indicated that differentially expressed miRNAs may contribute to the acquisition of radioresistance in cervical cancer. The expression of miRNA-320 was significantly changed in cervical cancer cells after radiation. In addition, β-catenin was highly expressed in C33AR cells compared with C33A cells at the protein level but not at the messenger RNA level (Fig. 2B and C).

Based on the differential expression of miRNA-320 between C33AR and its parental C33A cells, the potential role of miRNA-320 in cervical cancer radiobiology was examined by overexpressing or repressing miRNA-320 using synthetic miRNA-320 agomir/antiagomir in C33AR and C33A cells, respectively. The expression of miRNA-320 was tested by RT-qPCR (Fig. 3A and B). Following miRNA-320 overexpression, the survival rate of C33AR cells decreased compared with that of C33AR cells transfected with negative control vector 10 days after IR stimulation (Fig. 3C). These results revealed that overexpression of miRNA-320 significantly increases the sensitivity of C33AR cells to irradiation. The expression of miRNA-320 in C33A cells was inhibited by being transfected with antiagomir, and caused a decrease in radiosensitivity (Fig. 3D). In the proliferation assay, C33AR cells were transfected with miRNA-320 agomir and negative control, and exposed to IR with 4 Gy x-rays, and their cell growth was monitored by counting the cell numbers (Fig. 3E). Increased expression of miRNA-320 promoted cell death in the experimental group with 4 Gy IR exposure in the long-term cell culture compared with the negative control.

Hsieh et al have reported that miRNA-320 inhibits endogenous β-catenin expression and nuclear localization in prostate cancer (19). When β-catenin translocates to the nucleus, it activates the transcription of Wnt/β-catenin target genes, such as c-MYC and cyclin D1 (20). Therefore, to further explore the mechanism by which miRNA-320 influences cell radiosensitivity, the expression of β-catenin, c-MYC and cyclin D1 was examined by RT-qPCR and western blotting after transfecting C33AR cells with miRNA-320 agomir (Fig. 4A, B and D). Upon transfection with miRNA-320 antiagomir in C33A cells, the expression of β-catenin increased at the protein level (Fig. 4C). To confirm that miRNA-320 modulates the radiobiological behavior of cervical cancer cells by repressing β-catenin expression, rescue experiments were performed. First, β-catenin expression was inhibited using siRNAs (Fig. 4E) and then, the knockdown of β-catenin increased C33AR radiosensitivity (Fig. 4F). The inhibition of β-catenin rescued miRNA-320-mediated cell radioresistance. These results indicated that a decrease in miRNA-320 inhibits cervical cancer cell radiosensitivity in vitro by negatively regulating β-catenin expression and Wnt/β-catenin signaling activity.