Human uterine cervical cancer cells (HeLa) were cultured in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) and human colon cancer cells (HCT116) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, penicillin and streptomycin. The cells were maintained at 37°C in the presence of 5% CO₂. The cells were plated in T-25 flasks and treated with IR at various doses (0, 1, 3, 5, 8 and 12 Gy) following incubation at 37°C for 24 h to achieve logarithmic growth.

Synthetic miRNA mimics and siRNAs were obtained from GenePharma (Shanghai, China) (Table I). The HeLa and HCT116 cells were grown to 50% confluence in 6-well plates. The transfection of siRNAs and miRNAs was performed with jetPRIME (Polyplus, Illkirch, France) according to the manufacturer's instructions. The transfection medium was replaced with fresh medium after 6 h of incubation at 37°C. Total RNA and protein were prepared at 48 h following transfection and used for RT-qPCR detection or western blot analysis. The p38 and JNK agonist, anisomycin (Ani), the p38 inhibitor, SB203580 (SB), and the JNK inhibitor, SP600125 (SP), were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and dissolved in dimethyl sulfoxide (DMSO). The cells were respectively treated with Ani (20 µM), SB (10 µM), and SP (20 µM) for 1 h prior to IR and harvested for western blot analysis after 24 h of incubation at 37°C.

The 1,000, 800, 500 and 200 bp fragment of the genomic sequence immediately upstream of the miR-320a stem-loop structure were amplified by PCR from HeLa cell cDNA using Phusion DNA polymerase (Thermo Fisher Scientific). A pair of primers, including the XhoI and HindIII restriction sites were designed to amplify the miR-320a promoter inserts. The PCR products were cloned into the pGL3-enhancer vector (Promega, Madison, WI, USA).

Firstly, we searched the putative target sites of 3′UTR of XIAP by bioinformatics analysis using TargetScan (http://www.targetscan.org/) according to the instructions provided (http://www.targetscan.org/faqs.Release_7.html). The 3′UTR of XIAP was then cloned into a modified pGL3-control plasmid (21) following the PCR amplification of genomic DNA. The coding sequences of ATF2, ELK1 and YY1 were amplified from HeLa cell cDNA using a pair of primers, including the AscI and XhoI restriction sites and were ligated into pCMV6-AN-3DDK. The pCMV6-3DDK-XIAP vector was purchased from Origene (Rockville, MD, USA). The primer sequences are presented in Table I. The sequences of all constructs were confirmed by DNA sequencing.

Total RNA was extracted from the cells using TRIzol reagent (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. miRNA quantification was performed using a Quantitect SYBR-Green PCR kit (Qiagen, Hilden, Germany). Reverse transcription followed by RT-qPCR was carried out using a high capacity cDNA reverse transcription kit (Thermo Fisher Scientific) as previously described by Fu et al (22). Specific primer sequences were designed according to miRBase 22.0 (http://www.mirbase.org/). For pri-miRNA quantification, RT-qPCR was performed according to mRNA quantitation method (23). GAPDH was used as an endogenous control. All qPCR experiments were carried out using the Mx3000p detection system (Agilent Technologies, Palo Alto, CA, USA). The PCR thermal cycle began at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The primer sequences are presented in Table I. GenEX software version 5.0 was employed to analyze the qPCR data. One-way ANOVA with Dunnett's post hoc tests were performed to determine the difference in miRNA expression levels observed in the cells treated with IR at various doses (0, 1, 3, 5, 8 and 12 Gy) and treatment times (0, 3, 6, 12, 24, 48 and 72 h).

ChIP assays were carried out using a commercial kit from Cell Signaling Technology (CST, Danvers, MA, USA). Briefly, the cells were first fixed with 1% formaldehyde, and chromatin DNA was isolated with 1 µg anti-ATF2 (sc-187X), 1 µg anti-ELK1 (sc-355X), 1 µg anti-YY1 (sc-1703X), or 1 µg IgG (sc-2027) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) the indicated antibody and bound protein was digested with proteinase K. qPCR was performed using SYBR Premix Ex Taq (Takara, Dalian, China). The sequences of the primers used to amplify the ChIP samples are provided in Table I.

Total cell protein lysates were prepared using a modified radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate and 0.5% sodium dodecyl sulfate) in the presence of a proteinase inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were determined using BCA protein assay reagent (Beyotime, Shanghai, China). Protein samples (30 µg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon Hybond-C membranes (Amersham Biosciences, Piscataway, NJ, USA). After blocking with 5% non-fat dried milk for 3 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies. The antibodies used were as follows: ATF2 (1:1,000; ab32160), ELK1 (1:1,000; ab32106), YY1 (1:1,000; ab109237) (all from Abcam), XIAP (1:1,000; 2045), p-ATF2 (1:1,000; 9225), JNK (1:1,000; 9258), p-ELK1 (1:1,000; 9181), p-JNK (1:1,000; 4668), p38 (1:1,000; 9212), p-p38 (1:1,000; 4511), ERK (1:1,000; 4695), p-ERK (1:1,000; 4370) (all from Cell Signaling Technology) and β-actin (1:1,000; sc-47778; Santa Cruz Biotechnology). The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000; ZB2301; Zhongshan Jinqiao, Beijing, China). Signals were detected with the ECL western blot detection reagent (Thermo Fisher Scientific).

The HeLa cells were seeded in 60-mm flasks at 1×10⁵ cells, simultaneously transfected with a serial of RNA duplex and incubated at 37°C for a further 24 h. The cells were then treated with 8 Gy IR and incubated at 37°C for 24 h. The number of living cells was directly counted using the trypan blue exclusion method during the period of 5 days. Briefly, the cells were harvested and resuspended in 1 ml PBS. Subsequently, 1 part cell susupension was mixed with 1 part of 0.4% trypan blue (Beyotime). The mixture was incubated 3 min at room temperature and counted using a hemacytometer.

The HeLa cells were co-transfected with 100 ng. Firefly luciferase reporter vector containing plasmid constructs and 1 ng of a control vector containing Renilla luciferase pRL-TK (Promega) in a final volume of 0.5 ml RPMI-1640 in 24-well plates using jetPRIME. The Firefly and Renilla luciferase activities were measured consecutively using a dual-luciferase reporter assay system (Promega) at 48 h following transfection.

Annexin V apoptosis assays were performed using an Annexin V-EGFP kit (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions. The stained cells were immediately analyzed on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Each measurement was carried out at least in triplicate. The data were displayed as the percentage of apoptotic cells.

The cell proliferation rate was determined using cell growth curve analysis. Cells were seeded in 60 mm flasks with 1×10⁵ cells, simultaneously transfected with a serial of RNA duplex and incubated for a further 24 h. The cells were then treated with 8 Gy IR and incubated at 37°C for 24 h. The number of living cells was directly counted using the trypan blue exclusion method during the period of 5 days.

For colony forming unit (CFU) assays, the cells were transfected with miR-320a mimics, and 900 cells were seeded in each well of a fresh 6-well plate for 24 h prior to transfection. The CFU assay was performed 7 days after transfection, and the scoring criteria for colonies required at least 50 cells per colony.

Unless otherwise indicated, all the values are reported as the means ± standard deviation. Data analyses were estimated using SPSS software version 18.0 (IBM SPSS, Armonk, NY, USA). When the datasets contained multiple groups, statistical differences were evaluated by a one-way ANOVA with Dunnett's post hoc test. When only 2 groups were compared once in the datasets, statistical differences were evaluated using a Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

To identify which miRNAs are regulated by IR, we performed a miRNA microarray analysis in HeLa cells treated with 8 Gy IR. We previously found that the upregulation of miR-320a expression by IR was significant using a strict cut-off value of P<0.01 (>2.7-fold) (17). To confirm the microarray results, we analyzed miR-320a expression by RT-qPCR in the HeLa and HCT-116 cells under IR conditions. We found that the expression of miR-320a increased with time from 0 to 72 h in the HeLa cells (Fig. 1A) and HCT116 cells (Fig. 1B) treated with 8 Gy IR. In addition, the expression of miR-320a also increased with radiation doses ranging from 1 to 12 Gy at 24 h following exposure to IR (Fig. 1C) and with the radiation time ranging from 0 to 48 h following exposure to 8 Gy IR (Fig. 1D) in the HeLa cells. To determine whether miR-320a accumulation is mediated by transcriptional regulation, we also detected the expression of the primary precursor of miR-320a (pri-miR-320a). Of note, we found that pri-miR-320a exhibited similar expression profiles as miR-320a (Fig. 1E and F). Taken together, these data suggested that miR-320a was highly responsive to treatment with IR.

To elucidate the mechanisms through which miR-320a is upregulated by IR, we examined the precise location of the miR-320a promoter region. Four serial deletion luciferase reporter gene vectors (1,000, 800, 500 and 200 bp fragments) were constructed (Fig. 2A, top left panel), and their relative luciferase reporter units were measured. We observed that a 200 bp fragment was responsible for the robust induction of the miR-320a promoter. The analysis of the 200 bp miR-320a core-promoter sequence using the TFSEARCH program (http://diyhpl.us/~bryan/irc/protocol-online/protocol-cache/TFSEARCH.html) suggested that there were three potential transcription factor binding sites for ATF2, ELK1 and YY1. The mutation of any of these three binding sites could attenuate luciferase activities, and the simultaneous mutation of all three sites completely abolished the responsiveness of the promoter to these transcription factors (Fig. 2A, top right panel). Moreover, these three sites were highly conserved in the human, mouse, rat and dog miR-320a genomic sequences (Fig. 2A, bottom panel), implying that these elements were functionally important.

To determine whether these transcription factors directly bind the miR-320a promoter, we performed ChIP assays using anti-ATF2, anti-ELK1 and anti-YY1 antibodies. The results revealed that the miR-320a promoter sequences were enriched in these samples compared to the samples precipitated with an isotype-matched normal IgG. Moreover, an irrelevant DNA sequence (GAPDH coding region) was not specifically precipitated by these antibodies (Fig. 2B, top left panel). The ChIP results were also confirmed by RT-qPCR (Fig. 2B, bottom left and right panel). Moreover, we performed the ChIP assay using anti-ATF2 with a radiation time ranging from 0 to 48 h in HeLa cells (Fig. 2C) and HCT116 cells (Fig. 2D) treated with 8 Gy IR. The results indicated that the miR-320a promoter sequences were enriched by ATF2. Furthermore, the overexpression of ATF2, ELK1 and YY1 in HeLa cells could lead to the upregulation of pri-miR-320a expression (Fig. 2E). By contrast, the knockdown of ATF2, ELK1 and YY1 significantly suppressed the expression of pri-miR-320a (Fig. 2F). We obtained similar results in the HCT116 cells (data not shown). Taken together, these results suggest that ATF2, ELK1 and YY1 can bind the miR-320a promoter and promote the transcription of miR-320a under IR conditions.

Both the p38 MAPK and JNK signaling pathways play a vital role in cellular response to IR, UV light and oxygen stress (24,25). ATF2 and ELK1 can be both phosphorylated by p38 and JNK. In order to identify whether these signal transduction pathways stimulate miR-320a expression under IR conditions, we first examined the phosphorylation and activity of these two signal transduction pathways in response to IR in HeLa cells. As shown in Fig. 3A, the levels of phosphorylated p38 (p-p38) and phosphorylated JNK (p-JNK) were found to gradually increase from 3 to 48 h following treatment with 8 Gy IR, with a peak expression at 48 h; the total protein levels of p38 and JNK were not altered. By contrast, IR did not alter the phosphorylation or expression levels of endogenous ERK protein. As targets of p38 and JNK, the phosphorylation or expression levels of ATF2 and ELK1 were also activated (Fig. 3A); similar results were obtained with the HCT116 cells (data not shown). Moreover, these expression profiles were consistent with those of miR-320a expression (Fig. 1D).

To investigate whether the regulation of miR-320a expression by ATF2, YY1 and ELK1 is mediated by p38 and JNK, we detected their expression levels in HeLa cells treated with two specific chemical inhibitors of p38 (SB203580) and JNK (SP600125) or an agonist of p38 and JNK (anisomycin). The results revealed that either anisomycin or IR directly increased the protein expression or phosphorylation of a series of proteins, including p38, JNK, ATF2, ELK1 and YY1 (Fig. 3B, top panels). Moreover, the transcription of pri-miR-320a and miR-320a expression also increased (Fig. 3B, bottom panels). SB203580 or SP600125 inhibited the phosphorylation of ATF2 and ELK1, but not that of YY1 (Fig. 3B, top panels), resulting in the decreased expression of pri-miR-320a and miR-320a (Fig. 3B, bottom panels). The results suggested that IR induces the expression of YY1 and the phosphorylation of ATF2 and ELK to induce miR-320a expression through the p38 MAPK/JNK pathway.

miRNAs can regulate their target genes by inhibiting protein translation and/or degrading mRNAs. In this study, we found that the 3′UTR of XIAP had 3 predicted target sites, through TargetScan (http://www.targetscan.org/) (26). XIAP protein is necessary for anti-apoptotic function and is usually overexpressed in cancer cells (27,28).

In this study, we found that the overexpression or the inhibition of miR-320a had little effect on the mRNA level of XIAP in HeLa cells (Fig. 4A), but markedly affected its protein level (Fig. 4B). These results support the hypothesis that miR-320a regulates the expression of XIAP directly at the translational level.

To determine whether miR-320a targets the 3′UTR of the XIAP gene directly, luciferase reporter assays were performed using the HeLa cells. The results revealed that miR-320a significantly decreased the relative luciferase activity (~42%) of XIAP-3-3′UTR as compared to the negative control. However, the reporter with mutated effective target was not suppressed by miR-320a (Fig. 4C). These results indicated that XIAP was indeed a direct target of miR-320a.

To validate whether miR-320a induces apoptosis, we performed an Annexin V apoptosis assay using the HeLa cells. The results revealed that approximately 14.79% of HeLa cells transfected with miR-320a underwent apoptosis compared with 5.94% of untreated (mock) cells and 5.79% of the control cells. Similarly, 16.82% of the cells transfected with XIAP siRNA underwent apoptosis, suggesting that miR-320a promotes apoptosis by regulating XIAP expression (Fig. 5A).

IR can cause multiple types of cellular responses, such as DNA strand breaks, cell cycle arrest and apoptosis (29). Radiotherapy is an important treatment for cancer patients, and miR-320a expression can be induced by IR. This led us to examine whether miR-320a can affect the proliferation of tumor cells. We used the trypan blue exclusion method to evaluate the proliferation of HeLa cells overexpressing miR-320a. We observed that miR-320a inhibited cell proliferation compared with the negative control (Fig. 5B). In IR-treated HeLa cells, miR-320a also inhibited cell proliferation (Fig. 5B). When the miR-320a inhibitor was transfected into HeLa cells simultaneously, the HeLa cells exhibited a partial reversal of IR-induced apoptosis (Fig. 5C).

To determine the potential role of miR-320a in tumori-genesis, the colony formation capacity of the HeLa cells was evaluated following transfection with miR-320a. Notably, the miR-320a-overexpressing group displayed evidently fewer and smaller colonies compared with the control group (Fig. 5D). These data indicated that miR-320a enhanced radiosensitivity by inducing apoptosis and suppressing proliferation under IR conditions.

Overall, the results of this study suggested that IR induced the phosphorylation of p38 and JNK, and activated their downstream targets-ATF2, ELK1 and YY1, resulting in the transcription of miR-320a, thus increasing the expression of miR-320a to inhibit XIAP, and consequently inducing apoptosis and inhibiting proliferation to realize its biological function (Fig. 6).