The human tongue cancer cell lines SCC-15 and CAL27 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Cells were irradiated using the HL-2000 HybriLinker (UVP, Upland, CA, USA) at a wavelength of 254 nm. TSA was from Sigma-Aldrich (St. Louis, MO, USA), dissolved in dimethyl sulfoxide and stored at −80°C.

The chemically-modified double-stranded miR-375 mimic and the corresponding miRNA mimic control; a chemically-modified single-stranded miR-375 inhibitor and the corresponding miRNA inhibitor control were from RiboBio Co. (Guangzhou, China). The miR-375 mimic sequences were as follows: sense, 5′-UUU GUU CGU UCG GCU CGC GUG A-3′, and antisense, 5′-AAA CAA GCA AGC CGA GCG CAC U-3′; the miRNA mimic control sequences were: sense, 5′-UUU GUA CUA CAC AAA AGU ACU G-3′, and antisense, 5′-AAA CAU GAU GUG UUU UCA UGA C-3′; the miR-375 inhibitor sequence was: 5′-UCA CGC GAG CCG AAC GAA CAA A-3′; and the miRNA inhibitor control sequence was: 5′-UCU ACU CUU UCU AGG AGG UUG UGA-3′.

The cells were plated onto 6-well plates before transfection. After reaching 80% confluence, the cells were transfected with 30 nM miRNA mimic or 100 nM miRNA inhibitor using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Total RNA was extracted using the TRIzol reagent (Invitrogen) then reverse-transcribed into complementary DNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was conducted at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min using the ABI PRISM 7500 real-time PCR system (Applied Biosystems). The primer used for miR-375 reverse transcription was as follows: GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACT CAC GC; the primers for qRT-PCR of miR-375 were: sense, 5′-GTG CAG GGT CCG AGG T-3′, and antisense, 5′-AGC CGT TTG TTC GTT CGG CT-3′; and the primers for qRT-PCR of U6 (internal control for miRNA) were: sense, 5′-CTC GCT TCG GCA GCA CA-3′, and antisense, 5′-AAC GCT TCA CGA ATT TGC GT-3′. The data were analyzed using the 2^−∆∆Ct relative expression quantity as described previously (28).

Western blot analysis was performed as described previously (28). Briefly, cells were harvested, washed with PBS, and lysed in RIPA buffer. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Primary antibodies against phosphorylated AKT (p-AKT, Cell Signaling Technology, Beverly, MA, USA), AKT (Cell Signaling Technology), PDK1 (Cell Signaling Technology), and β-actin (Cell Signaling Technology) were diluted at 1:1,000. The intensities of the bands were quantified using ImageJ software (http://rsb.info.nih.gov/ij/). The background was subtracted, and the signal of each target band was normalized to that of the β-actin band.

Cell proliferation was measured using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Briefly, the cells were plated onto 96-well plates (2×10³ cells/well). After treatment with TSA and/or exposure to a specific dose of radiation, CCK-8 (10 µl) was added to each well at various time points and incubated at 37°C for 3 h. The absorbance at 450 nm was measured using a microplate spectrophotometer (Bio-Tek Instruments Inc., Winosski, VT, USA).

CAL27 cells (1×10³ cells/well) seeded in 6-well plates were treated with TSA, and then exposed to the indicated dose of radiation. The cells were cultured for 2–4 weeks, and the colonies were detected by staining with crystal violet (0.5% in 20% ethanol). Next, cells were harvested and measured by a multifunctional micro-plate reader at 546 nm. The relative colony number (relative survival cell number) = OD 546 administration group/OD 546 control group, and the radiation survival curve was drawn.

The cells were cultured on coverslips and treated with TSA or transfected with miR-375 mimics. Next, the cells were exposed to a specific dose of radiation. Then the cells were subjected to TUNEL staining (one-step TUNEL Apoptosis Assay kit, Beyotime, Jiangsu, China) according to the manufacturer's protocol. Briefly, cells were fixed in 4% paraformaldehyde, treated with 0.1% Triton X-100, and labeled with fluorescein-12-dUTP using terminal deoxynucleotidyl transferase. Cell nuclei were stained with DAPI. All fluorescent images were examined using a Leica DM3000 microscope (Leica, Solms, Germany).

The cellular enzymatic activity of caspase-3 and −7 was determined using a colorimetric assay (Caspase-Glo 3/7 Assay Systems, Promega, Madison, MI, USA). Briefly, for each reaction, cells were lysed and incubated with a luminogenic substrate containing the DEVD sequence, which is cleaved by activated caspase-3/7. After incubation at room temperature for one hour, luminescence was quantified using a Centro XS3 LB 960 luminometer (Berthold, Bad Wildbad, Germany).

The acetylation of miR-375 promoter was assessed using ChIP, which was performed using an EZ-Magna ChIP assay kit (Merck Millipore, Darmstadt, Germany). Briefly, we sonicated the crosslinked chromatin DNA to generate 200–1,000 bp DNA fragments. DNA-protein complexes were immunoprecipitated with specific antibodies against acetyl-histone H3 (ac-H3). Normal IgG was used as the negative control. The protein/DNA complexes were then eluted and reverse cross-linked. Spin columns were used to purify the DNA. The precipitated DNA was quantified by qPCR. Relative enrichment was calculated as the amount of amplified DNA normalized to the input and relative to values obtained after normal IgG immunoprecipitation. The primer specific for human GAPDH was used as positive control. The sequences are as follows: sense, 5′-TAC TAG CGG TTT TAC GGG CG-3′, and antisense, 5′-TCG AAC AGG AGG AGC AGA GAG CGA-3′. The primer sequences for ChIP-1 are as follows: sense, 5′-ACT ACA TCG CCT GGG TTT GA-3′, and antisense, 5′-TGC CAA ATA TGC TGC TGG TT-3′; the ChIP-2 sequences are: sense, 5′-CAC CGC CAG TAA AAG CAT CT-3′, and antisense, 5′-CCA TCC TTC CCT CTC AGA GC-3′.

Statistical analyses were performed using SPSS 16.0 (SPSS, Chicago, IL, USA). The data are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Differences between groups were analyzed using Student's t-test. In cases of multiple-group testing, one-way analysis of variance (ANOVA) was used. A two-tailed value of P<0.05 was considered statistically significant.

To determine the dose of irradiation and TSA that could be used without significantly affecting cell vitality, TSCC cells were treated with different concentrations of TSA or different dose of irradiation. Neither TSA (1 µg/ml) nor irradiation (4 Gy) significantly affected the vitality of TSCC cells (Fig. 1A), so we used these non-cytotoxic doses in the subsequent experiments. We then used CCK-8 assays to determine whether TSA can regulate radiosensitivity in TSCC cells. Treatment with TSA or irradiation alone only slightly reduced the cell proliferation until 72 h after treatment. However, combination treatment with TSA and irradiation significantly reduced the proliferation of SCC-15 and CAL27 cells from 24 to 72 h after treatment (Fig. 1B). The colony formation ability of CAL27 cells was also examined. The results showed that after a mild dose of irradiation treatment (≤6 Gy), the colony formation of CAL27 cells was gradually attenuated (Fig. 1A), and pretreatment with TSA (1 µg/ml) further enhanced the activity of irradiation (Fig. 1A).

To further confirm that TSA increases the radiosensitivity of TSCC cells, we measured caspase-3/7 and apoptosis after the treatments. The activity of caspase-3/7 was upregulated after treatment with TSA or irradiation alone, and further upregulated after the combinational treatment with TSA and irradiation (Fig. 2A). Consistently, the number of apoptotic cells revealed by TUNEL staining was slightly increased by treatment with TSA or irradiation alone, and was significantly increased by the combinational treatment with TSA and irradiation (Fig. 2B).

To determine whether miR-375 is involved in the regulation of radiosensitivity by TSA, we first treated the TSCC cells with TSA in different concentrations (0.5–2 µg/ml) and time (24–72 h), and found that TSA upregulated miR-375 expression dose-dependently (Fig. 3A) and time-dependently (Fig. 3B). Then, we checked the histone acetylation in the miR-375 promoter region by ChIP assays after TSA treatment. Two pairs of primers were designed to analyze the status of ac-H3 in the promoter. We found that TSA significantly increased the levels of ac-H3 in the fragment containing ChIP-1 and ChIP-2 (Fig. 4A), suggesting that restoration of histone acetylation in the promoter region induces miR-375 expression after TSA treatment. Further, we found that the level of AKT phosphorylation was reduced after TSA treatment in TSCC cells, and overexpression of miR-375 inhibited the expression of PDK1, followed by the suppression of AKT phosphorylation without significant change of total AKT levels (Fig. 4B).

To determine whether miR-375 overexpression could increase radiosensitivity of TSCC cells, SCC-15 and CAL27 cells were transiently transfected with a control miRNA or miR-375 mimic, and their radiosensitivity was determined. The TSCC cells treated with irradiation or transfected with the miR-375 mimic did not significantly change the percentage of apoptotic cells compared with the control. However, combining overexpression of miR-375 with irradiation induced significant TSCC cell apoptosis compared with the control as revealed by the activity of caspase-3/7 (Fig. 5A) and TUNEL staining (Fig. 5B).

To determine whether TSA-induced radiosensitivity is mediated by miR-375, an inhibitor specific for miR-375 was used to analyze the effects of miR-375 knockdown on the cellular radioresponse. The inhibition of cell proliferation induced by combining TSA with irradiation was relieved by miR-375 knockdown (Fig. 6A), and the apoptosis induced by the combined treatment was reduced by miR-375 knockdown as revealed by the activity of caspase-3/7 (Fig. 6B) and TUNEL staining (Fig. 6C).