The human cervical cancer cell lines Siha and C33A and mouse fibroblast cell line NIH3T3 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Rockville, IN, USA). Rabbit anti-E-cadherin, anti-β-catenin, anti-vimentin, anti-NF-κB p65 antibody and phospho-NF-κB p65 (Ser536) (93H1) rabbit mAb were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (H+L) antibody and HRP-labelled goat anti-mouse IgG (H+L) antibody were obtained from Kirkegaard & Perry Laboratories, Inc. (KPL, Gaithersburg, MD, USA). Cell lysis buffer for western blotting was purchased from Biocolor BioScience & Technology Co. (Shanghai, China).

The human cervical cancer cell lines Siha and C33A were cultured in MEM supplemented with 10% FBS and the mouse fibroblast cell line NIH3T3 was cultured in DMEM supplemented with 10% FBS and 100 U of penicillin-streptomycin at 37°C in a humidified 5% CO₂ incubator.

The cervical cancer cells Siha and C33A cells were cultured in 25-cm² flasks. When the cells reached 75% confluence, they were treated using the Faxitron Cabinet X-ray System (Faxitron, Wheeling, IL, USA) as previously described (18). The irradiated cells were derived after irradiation of the parental cells at a dose rate of 0.36 Gy/min (given in 2-Gy fractions five times/week) with a cumulative dose of 75 Gy. The control cells were treated using the same procedure except that they were unirradiated. The culture medium of the parental cells and X-ray-irradiated cells was changed every 3 days and the cells were passaged every 7 days. After reaching 75 Gy, the surviving cells were allowed to rest and reorganise for 2 weeks before experiments.

Total RNA was isolated using TRIzol reagent according to the manufacturer’s protocol and reverse transcriptase reactions were performed using 1 μg total RNA and the PrimerScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). For real-time PCR, the Applied Biosystems StepOnePlus™ Real-Time PCR System was used according to the manufacturer’s protocol. The relative gene expression ΔCt values of mRNA were calculated by normalising to expression of the endogenous control GAPDH. Each experiment was repeated three times.

The cells were washed three times with PBS, scraped and lysed in RIPA buffer (Shennengbocai, Shanghai, China) (0.1% SDS, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate and 1 mM sodium vanadate) containing protease inhibitors (1 mM sodium fluoride, 1 mM PMSF). The lysates were harvested on ice for 30 min and centrifuged at 12,000 rpm for 15 min at 4°C. The supernatants were collected and the protein content was measured using the BCA Protein Assay kit (Merck, Darmstadt, Germany). Seventy micrograms of total protein was separated by 5–10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore) using a semi-dry blotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% (w/v) non-fat milk in TBST (100 mM NaCl, 50 mM Tris and 0.1% Tween-20) at room temperature for 1 h and then they were incubated overnight with primary antibodies at 4°C. After washing three times with TBST, the membranes were incubated with appropriate HRP-labelled secondary antibodies (KPL) for 2 h. Finally, the signals of protein bands were detected with an ECL system (Merck) according to the manufacturer’s instructions. β-actin was used as the endogenous loading control.

For the migration assays, the lower chamber was filled with 500 μl of conditional medium from NIHT3 cells. The cells were digested with trypsin and 1×10⁵ cells suspended with serum-free MEM were seeded onto the upper chamber. After 16 h, the non-migrating cells inside the filters were removed with a cotton swab and the migrating cells on the other side of the filter were fixed with methanol and stained with 0.1% crystal violet. The number of cells was counted in at least 10 random fields under a light microscope and data were analysed statistically.

For the invasion assays, a Transwell system (8-μm pore size; BD Biosciences, Bedford, MA, USA) precoated with 2 mg/ml of basement membrane Matrigel (BD Biosciences) was used. The cells were treated similar to the procedure used for the migration assay described above. After 24 h, the filters were fixed and stained similar to the procedure used for the migration assay. Experiments were performed in triplicate.

For the RNA interference assay, Lipofectamine 2000 (Invitrogen) was used according to the manufacturer’s instructions. The cells were prepared at a density of 2×10⁶ cells/flask and washed twice with PBS; then, a transfection mixture of NF-κB p65 siRNA was added to the flasks. After a 4-h incubation at 37°C in a 5% CO₂ in a humidified incubator, the transfection medium was discarded and replaced with complete medium.

Statistical analysis was performed using Student’s t-test. Results are presented as the mean ± standard deviation (SD). P<0.05 was considered statistically significant and the experiments were repeated at least three times.

When the treatment of parental cervical cancer cells with low-dose X-ray radiation reached the total of 20-Gy, the morphology of cells changed from a polarised epithelial cell type to a mesenchymal cell type. As shown in Fig. 1, the parental cells exhibited a typical cobblestone-like shape and tight cell junctions, which are traits of the epithelial phenotype. Additionally, the FIR cells treated with low-dose X-ray radiation demonstrated a spindle-like, elongated phenotype with loss of cell-to-cell contact, features that are characteristic of fibroblasts.

Preview results showed that the low-dose radiation treatment induced the morphological changes of cells. To evaluate if this change was the result of epithelial-mesenchymal transition (EMT), we tested the expression of EMT markers in FIR cells and their parental cells. Real-time qPCR results showed that expression of the epithelial markers E-cadherin and CK-18 were remarkably downregulated (P<0.01) and the mesenchymal markers N-cadherin and vimentin were upregulated in FIR cells. Additionally, the expression of N-cadherin in Siha FIR cells was highly significantly upregulated (P<0.01, P=0.00066) and the expression of vimentin in the two FIR cell lines was significantly upregulated compared with that of their parental cells (P<0.05) (Fig. 2A). Next, we determined the protein expression levels of the two FIR cell lines. Western blot analysis was consistent with these results. The expression of E-cadherin was decreased and N-cadherin and vimentin expression were increased compared with their parental cells (Fig. 2B). These results indicated that low-dose irradiation in cervical cancer cells induced EMT.

Cells that undergo EMT have demonstrated increased migration and invasion ability (1). To determine whether these FIR cells display such traits, we evaluated the migration and invasion ability of FIR cells using the two-chamber assay. As shown in Fig. 3, the migration ability of the two FIR cell lines (P<0.01) was significantly enhanced compared with that of their parental cells (P<0.05). The 16-h invasion assay indicated that the two FIR cell lines acquired a stronger invasive ability than their parental cells (Fig. 3B).

NF-κB was reported to be involved in Snail gene-regulated EMT (19,20). Additionally, our lab previously demonstrated that the p65 subunit of NF-κB plays a role in MTDH-induced EMT (21). To determine whether the p65 subunit of NF-κB was involved in the EMT process induced by low-dose radiation, we evaluated the expression of p65 and phosphorylated p65 by western blot analysis in parental and FIR cervical cancer cells. As shown in Fig. 4A, p65 expression was upregulated and phosphorylated p65 expression was downregulated in the two FIR cell lines. We then further tested the roles of NF-κB in low-dose radiation-induced EMT by transfecting NF-κB p65 siRNA into FIR cells. The RNA interference efficiency of p65 was tested by western blot analysis 48 h after transfection (Fig. 4B). Real-time qPCR results showed that the miRNA expression of E-cadherin and CK-18 was upregulated and that of N-cadherin and vimentin was downregulated after transfection with NF-κB p65 siRNA (Fig. 4C). After transfection with NF-κB p65 siRNA, E-cadherin expression was significantly upregulated (P<0.01) and the expression of N-cadherin was downregulated (P<0.05). The results of western blot analysis of the EMT markers were consistent with the real-time qPCR results (Fig. 4D). These results suggested that p65 was involved in EMT induced by low-dose radiation.