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Introduction

Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer-associated mortalities among females worldwide (1). Most of these cases and deaths occurred in developing countries (2). Despite showing a high cure potential when diagnosed early, cervical cancer continues to be a significant health threat to females and remains a serious issue of public health concern, especially in developing countries. Cervical cancer is usually treated with surgery, radiotherapy and chemotherapy depending on the stage of disease. Radiotherapy serves as the standard adjuvant approach in the management of cervical cancer across different clinical sub-types. For some early stage patients, radiotherapy can achieve the same effect as radical surgery (3). However, in clinical practice, some patients often develop resistance to the radiation treatment, resulting in treatment failure and eventually poor outcomes. Thus, it is important to improve radiosensitivity in those patients and explore the mechanisms underlying radioresistance (4).

MicroRNAs (miRNAs) are a type of endogenously expressed small, non-coding RNA sequences (~22 nucleotides) with a role in gene expression regulation at the post-transcriptional level (5). miRNAs bind to the 3′-untranslated regions (UTRs) of their target mRNAs and repress the translation or stability of target mRNAs (6). By downregulating the oncogenes or tumor suppressors, miRNAs may play tumor suppressive or oncogenic roles. miRNAs can alter various cell processes, including developmental transitions, organ morphology, bilateral asymmetry, stress response, metabolism, cell proliferation, apoptosis and the response to radiation (5,7,8). In recent years, the relationship between miRNAs and the radiosensitivity of different cancers has been identified (Table I). Those studies showed that miRNAs are potentially useful in manipulating the radiation response in the clinic to enhance susceptibility to or protect cells from radiation. In human cervical cancer, the expression of miR-18a was significantly decreased in the radioresistant tumor samples as compared with that in the radiosensitive tumor samples as identified by microarray analysis (9).

Table I Differential expression of microRNAs in various types of cancer and the effect on sensitivity to radiotherapy.

Table I

Differential expression of microRNAs in various types of cancer and the effect on sensitivity to radiotherapy.

MicroRNA	Cancer type	Target gene(s)	Effect on sensitivity to radiotherapy	Refs.
hsa-let-7 family	Lung cancer	RAS	Up/Down	(33)
hsa-miR-521	Prostate cancer	CSA	Up	(34)
hsa-miR-221/222	Gastric cancer	PTEN	Down	(35)
hsa-miR-221	Colorectal carcinoma	PTEN	Down	(36)
hsa-miR-21	Esophageal cancer	PTEN	Down	(37)
hsa-miR-181a	Malignant glioma	Bcl-2	Up	(38)
hsa-miR-34	NSCLC	p53	Up	(39)
hsa-miR-148b	NHL	DNMT3b	Up	(40)
hsa-miR-9	Lung cancer	NF-κB1	Up	(41)
hsa-miR-218	Cervical cancer	Rictor protein	Up	(4)
hsa-miR-155	Lung cancer	FOXO3A	Down	(42)
hsa-miR-29c	NPC	Bcl-2 and Mcl-1	Up	(43)
hsa-miR-18a	Breast cancer	ATM	Up	(13)

[i] NSCLC, non-small cell lung cancer; NHL, non-Hodgkin’s lymphoma; NPC, nasopharyngeal carcinoma; CSA, Cockayne syndrome protein A; ATM, ataxia-telangiectasia mutated.

In the present study, we revealed that the mean expression level of miR-18a was significantly higher in radiosensitive patients compared with radioresistant patients. Moreover, we demonstrated that miR-18a re-sensitizes cervical cancer cells to radiotherapy by impairing DNA damage repair and promoting cell apoptosis. We proved that miR -18a is a potential predictor of radiosensitivity and a therapeutic target in cervical cancer patients.

Materials and methods

Patient characteristics and tissue specimens

Primary cervical carcinoma tissues were collected from Wuhan Union Hospital. The samples were obtained after written informed consent was provided by the patients. The study was approved by the Ethics Committee of Wuhan Union Hospital of Tongji Medical College of Huazhong University of Science and Technology. A total of 48 clinical tissue samples were verified by histopathological examination and stored at −80°C prior to use. The tumor samples were obtained from the patients who did not receive any previous therapy. The samples were classified into two groups according to the response to radiotherapy: the radiosensitive group included patients who achieved complete response (CR) after radical radiotherapy, while the radioresistant group included patients who achieved partial response (PR) or relapsed within six months. The patient characteristics are shown in Table II.

Table II Different clinicopathological characteristics of cervical cancer patients.

Table II

Different clinicopathological characteristics of cervical cancer patients.

Clinicopathological characteristics	All cases	Radiotherapy response		P-value
		Radioresistant (%)	Radiosensitive (%)
Age (years)
<50	23	8 (34.8)	15 (65.2)	0.353
≥50	25	12 (48)	13 (52)
Histological classification
Squamous cell carcinoma	44	16 (36.4)	28 (63.6)	0.013
Adenocarcinoma	4	4 (100)	0
FIGO stage
I-IIb	34	15 (44.1)	19 (55.9)	0.591
IIIa-IV	14	5 (35.7)	9 (64.3)
Tumor size (cm)
<4	21	8 (38.1)	13 (61.9)	0.658
≥4	27	12 (44.4)	15 (55.6)

Cell culture

The SiHa and HeLa human cervical cancer cell lines were provided by the Gynaecology and Obstetric Laboratory of Wuhan Union Hospital. SiHa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and supplemented with 10% fetal bovine serum (FBS) (both from Hyclone, Logan, UT, USA). HeLa cells were routinely cultured in RPMI-1640 medium containing 10% FBS (Hyclone). The cell lines were maintained in a humidified incubator at 37°C in a 5% CO2 atmosphere.

Transfection

The cells (2×105 cells/well) were seeded in 6-well plates and transfection was carried out 24 h later using Opti-MEM and Lipofectamine 2000 (Invitrogen, Life Technologies) according to the manufacturer’s instructions until the cells reached a ~30–50% confluence. The miR-18a mimic, inhibitor and their negative controls were purchased from RiboBio (Guangzhou, China). miR-18a expression was increased or decreased in the SiHa and HeLa cells by transient transfection with miR-18a mimic at a final concentration of 50 nM or miR-18a inhibitor at a final concentration of 100 nM. The negative control was included in the parallel experiment. Following transfection, the cells were subjected to further assays or for RNA/protein extraction.

RNA extraction, reverse transcription and quantitative PCR

Total RNA was isolated from the transfected cells and human tissues using TRIzol reagent (Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. miR-18a expression was assessed using SYBR-Green quantitative reverse transcription (RT)-PCR (Takara Biotechnology, Dalian, China) with U6 snRNA as a normalizing control on a StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The cycling conditions were: 95°C for 30 sec, 60°C for 5 sec, 95°C for 15 sec, 60°C for 1 min and 95°C 15 sec, which were repeated for 40 cycles. The gene expression was measured using the following primers: miR-18a, (forward), 5′-GTTCCTAAGGTTCATCTAGTGCAGATA-3′ and (reverse), 5′-CAGTGCAGGGTCCGAGGTAT-3′; U6, (forward), 5′-CTCGCTTCGGCAGCACATA-3′ and (reverse), 5′-CGAATTTGCGTGTCATCCT-3′. The relative expression of miR-18a was calculated using the relative quantity (RQ) 2−ΔCT (in tissues) or 2−ΔΔCt (in cells) method against U6 for normalization, where ΔCT = (CTmiR-18a-CTU6snRNA), ΔΔCT = (CTmiR-18a-CTU6snRNA) mimic/inhibitor - (CTmiR-18a-CTU6snRNA) control. Each sample was analyzed in triplicate.

MTT assay

The MTT assay was used to detect the proliferation rate of the cervical cancer cells following transfection. The cells (5×103 cells/well) were seeded in a 96-well plate for 24 h before transfection and then allowed to grow in normal medium. For the MTT assay, the cells were incubated in 20 μl MTT (5 μg/ml) at 37°C for 4 h and lysed in 150 μl dimethyl sulfoxide (DMSO) at room temperature for 10 min with slight agitation. The absorbance in each well was measured at 490 nm (test wavelength) and 630 nm (reference wavelength) using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The experiment was repeated three times and six parallel samples were measured each time.

Hoechst staining

The transfected cells were fixed with 10% absolute methanol for 10 min, and washed twice with PBS. Then cells were stained with Hoechst 33342 (Beyotime, Shanghai, China) for 15 min in the dark and then washed twice with PBS. The nuclear morphology of the cells was visualized using an inverted fluorescence microscope. The cells showing nuclear fragmentation and chromatin condensation in Hoechst staining were considered apoptotic cells (10). Three independent experiments were performed in triplicate.

Western blot analysis

The parental and transfected cells were exposed to 8 Gy X-rays, and then the cells were collected and lysed 24 h after irradiation. Protein was extracted from the lysates and the concentrations was measured using the BCA Protein Assay system (Beyotime). Anti-ATM, anti-γ-H2AX, anti-caspase-3 and anti-PARP antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin protein levels were used as a control to verify equal protein loading.

Clonogenic cell survival in vitro

The SiHa and HeLa cells (2×105 cells/well) were seeded in 6-well plates 24 h before transfection. After 48 h of transfection, the cells were seeded in culture dishes and irradiated the following day at distinct doses (0, 2, 4, 6 and 8 Gy). The plates were incubated at 37°C in a 5% CO2 atmosphere for 12 days. The cells were fixed with 4% paraformaldehyde and then stained with 1% crystal violet, and colonies containing ≥50 cells in size were counted. The survival curve was derived from the multi-target single-hit model: SF = 1-(1-e−D/D0)N using the multi-target click model of GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). The sensitization-enhancing ratio (SER) was then calculated (a ratio of SF2).

Flow cytometric analysis of apoptosis

Transfected cells were irradiated with a dose of 8 Gy. The cells were collected and resuspended 24 h after ionizing radiation (IR) according to the manufacturer’s instructions (Beyotime). Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining assays were conducted to detect the percentage of apoptotic (FITC-stained) and necrotic (PI-stained) cells in a given population. Analyses were performed by a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), the procedures were repeated in triplicate.

Immunofluorescent staining

Transfected cells were exposed to 2 Gy X-rays, an appropriate dose according to most radical radiotherapy protocols (11). The cells were collected and stained with anti-γ-H2AX (Cell Signaling Technology) antibody at different times as previously described (12). Images were captured using a laser scanning confocal microscope (Olympus Optical Co., Tokyo, Japan). Red showed γ-H2AX foci and blue nuclei stained with DAPI. For each treatment, a γ-H2AX foci number of ≥300 cells was counted.

Statistical analysis

Statistical analysis was performed with the SPSS 21.0 statistical software package (SPSS, Chicago, IL, USA). The results were shown as mean ± SD. Differences in measured variables between experimental and control groups were assessed using the Student’s t-test. The receiver operating characteristics (ROC) curve analysis was used to investigate the predictive value of miR-18a expression in cervical cancer radiotherapy response. The Mann-Whitney U test was performed to compare the differences in the miR-18a expression levels between radiosensitive and radioresistant patients. In all cases, P<0.05 was considered to indicate a statistically significant difference.

Results

Detection of high level of miR-18a expression is in radiosensitive patients

To determine whether miR-18a was associated with radiosensitivity in cervical cancer, we first compared the miR-18a expression level in radiosensitive and radioresistant patients. Results are shown in Fig. 1 after normalization to control U6 expression. We detected that the mean expression level of miR-18a was significantly higher in radiosensitive than in radioresistant patients by RT-qPCR. This result revealed that miR-18a had, a linear correlation with radiosensitivity.

Figure 1 Scatter plots of miR-18a expression in cervical cancer patients. The expression level of miR-18a is significantly higher in radiosensitive than in radioresistant patients as shown by RT-qPCR. The line is the median value (Log10 scale at y-axis, *P<0.05).

Tissue miR-18a as a potential diagnostic marker for the sensitivity of radiotherapy

The ROC curve and the area under the curve (AUC) were used to assess the feasibility of using miR-18a expression level as a diagnostic tool for detecting radiosensitivity. The data using a ROC curve showed obvious separation between the two groups, with an AUC of 0.814 and a 95% CI (confidence interval) of 0.685–0.944 (Fig. 2). At the cut-off value of 6.49×10−6 (relative expression in comparison with U6), the sensitivity was 89.3% and the specificity was 70%. This result indicated that miR-18a is a potential molecular marker for the detection of the sensitivity to radiotherapy.

Figure 2 ROC curve analysis in the miR-18a assay for detecting radiosen-sitivity. The AUC is 0.814 (95% CI, 0.685–0.944). ROC, receiver operating characteristic; AUC, area under the ROC curve; CI, confidence interval.

Overexpression of miR-18a increases sensitivity of cervical cancer cells to radiation treatment

To determine the effect of miR-18a on the sensitivity of cervical cancer cells to radiotherapy, we transfected miR-18a mimic or inhibitor into the SiHa and HeLa cells. A qPCR analysis showed that miR-18a was markedly upregulated or downregulated when transfected with miR-18a mimic or inhibitor, particularly 48 h post-transfection (Fig. 3). Results of the clonogenic assay showed the surviving fractions (SF) at each dose (2, 4, 6 and 8 Gy) decreased in miR-18a overexpressed cells (Fig. 4). The values of D0, Dq and SF2 were lower in the miR-18a-upregulated cells than the control and blank groups (Table III). Therefore, we concluded that the miR-18a increased the cellular sensitivity to radiation treatment in vitro.

Figure 3 Expression of miR-18a in cells transfection with miR-18a mimic or inhibitor. Cells (2×105 cells/well) were seeded in 6-well plates and transfected 24 h later. Then, we extracted the RNA from transfected cells at different times (d1, d2, d3, d6, d9, d12). qPCR analysis shows that miR-18a was markedly upregulated or downregulated post-transfection (**P<0.01, ***P<0.001).

Figure 4 Clonogenic assay showing that miR-18a sensitizes cervical cancer cells to irradiation. Various tumor cells (for SiHa, the numbers were 200, 500, 1,500, 4,000 and 8,000; for HeLa, the numbers were 200, 500, 1,500, 8,000 and 12,000) were plated in 6-well plates according to different doses before irradiation. Colonies containing ≥50 cells were counted 12 days later. Survival curves show data fitting according to the multi-target single-hit model: SF =1-(1-e−D/D0)N (SF, cell survival fraction; D, radiation dose; e, the bottom of the natural logarithm; D0, the mean death dose; N, extrapolate number). Data are shown as the mean ± SD from three independent experiments.

Table III Main parameters of cell survival curves following irradiation.

Table III

Main parameters of cell survival curves following irradiation.

Parameters	Mock		Mimic-18a NC		Mimic-18a
	SiHa	HeLa	SiHa	HeLa	SiHa	HeLa
D0	2.04	2.66	2.13	2.58	1.66	1.67
Dq	4.92	3.10	3.56	2.44	1.38	0.94
N	3.41	2.17	2.68	1.95	1.83	1.56
SF2	0.79	0.73	0.71	0.68	0.47	0.43

[i] D₀, mean lethal dose; D_q, quasi-threshold dose; N, extrapolation number; SF₂, surviving fraction at 2 Gy.

Overexpression of miR-18a enhances radiation-induced apoptosis

To investigate the effect of miR-18a on the sensitivity of cervical cancer cells to radiotherapy, we examined cell apoptosis following treatment of irradiation with a flow cytometry assay. We detected that miR-18a enhanced the cellular sensitivity to radiation by promoting cell apoptosis following exposure to 8-Gy irradiation. As shown in Fig. 5, the percentage of apoptotic cells was significantly increased in the miR-18a mimic-transfected cells than the control cells, but decreased in the miR-18a inhibitor transfected cells than the control cells. What is more, apoptosis was detected through an increase of cleaved caspase-3 and PARP by western blotting, which were two classic characteristics of cell apoptosis. Western blot analysis demonstrated that 24 h after exposure to 8-Gy irradiation, cleaved caspase-3 and PARP protein expression in the SiHa and HeLa cells transfected with miR-18a mimic were higher than that in cells transfected with a control (Fig. 6).

Figure 5 miR-18a enhanced radiation-induced apoptosis. Cells were exposed to 8-Gy irradiation and harvested 24 h later. Apoptosis was determined by flow cytometry. FITC-stained positive cells were regarded as apoptotic cells. Data in the right panels show the apoptotic ratios, and a significant difference was determined in the transfected groups compared with the control groups (*P<0.05).

Figure 6 After treatment with 8 Gy irradiation, the cleavages of caspase-3 and PARP were detected by western blotting. The results were consistent with those of the flow cytometry assays.

miR-18a expression has no effect on the proliferation and apoptosis of cervical cancer cells

To determine the biological role of miR-18a in cervical cancer, an MTT assay was performed to examine the relationship between miR-18a expression and cell proliferation. Consequently, ectopic upregulation or downregulation of miR-18a had no significant impact on the proliferative activity of the SiHa and HeLa cells compared with the control groups (Fig. 7). In the Hoechst staining assay, the nucleus which exhibited brightly stained condensed chromatin, indicated typical morphological changes of apoptosis. In this experiment, the number of Hoechst-positive apoptotic cells had no difference in each group (Fig. 8). These data showed that miR-18a had no effect on the proliferation and apoptosis of cervical cancer cells without irradiation treatment.

Figure 7 MTT assay showing that upregulated or downregulated miR-18a in the SiHa and HeLa cells had no impact on the proliferative activity (P>0.05). Data are demonstrated as absorbance. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 8 Hoechst staining assay was performed after transfection and the result shows that the expression of miR-18a had no effect on apoptosis in the SiHa and HeLa cells (P>0.05).

MiR-18a suppresses ATM expression by targeting 3′-UTR of ATM

miRWalk, GenBank, TargetScan and mirBase were used to predict the target genes of miR-18a to determine how miR-18a modulates the sensitivity of cancer cells to radiation. Analysis with these algorithms, revealed that ATM was a potential target of miR-18a. In addition, the luciferase assay, as previously reported, demonstrated that miR-18a specifically affected the ATM-3′-UTR (13,14). It has been previously reported that radiation treatment upregulated ATM expression, leading to phosphorylation of the repair proteins (15). Therefore, we examined whether miR-18a enhanced the radio-sensitivity of cervical cancer cells by targeting ATM using western blotting after exposure to 8 Gy irradiation. The results showed that the level of ATM protein was significantly down-regulated following miR-18a overexpression but upregulated when miR-18a was suppressed. Results of downstream protein, γ-H2AX, were similar to those of ATM (Fig. 9).

Figure 9 Western blotting shows that the level of ATM protein was significantly downregulated following miR-18a overexpression (A) but upregulated when miR-18a was suppressed (B). The downstream protein, γ-H2AX, had similar results to ATM after exposure to 8 Gy. β-actin was used as the loading control. ATM, ataxia-telangiectasia mutated.

Overexpression of miR-18a impairs the double-strand break (DSB) repair induced by radiation

One of the most important therapeutic functions of radiation is to induce DNA DSB in cancer cells. When cells respond to DNA damage, the initial step is the phosphorylation of repair proteins, such as the ATM and H2AX (11,16,17). If the ATM decreases, the DSB repair signaling pathway is impaired and cell apoptosis is enhanced. We monitored the kinetics of the DSB repair process by immunofluorescent staining of γ-H2AX foci at different time-points post-irradiation to 2 Gy. As shown in Fig. 10, a rapid increase of the number of γ-H2AX foci was evident following irradiation, reaching a peak at 30 min. The level of fluorescence then decreased significanly as the DSB repair was completed. Overexpression of miR-18a downregulated the ATM and reduced DNA repair, while inhibition of miR-18a led to an increase of DNA damage repair by upregulated ATM. The change of γ-H2AX foci was consistent with the western blot results, suggesting that the reduction of ATM led to a low efficiency of DSB repair as we hypothesized. These results further corroborated the role and mechanism of miR-18a in enhancing cancer cell radiosensitivity to radiotherapy.

Figure 10 miR-18a impairs the ATM signaling pathway. Cells were treated with IR (2 Gy), and immunostained with γ-H2AX antibody at different time-points after radiotherapy. Left panel shows the micrographs of γ-H2AX foci formation after IR treatment, and the right panel shows the quantification of IR-induced γ-H2AX. More than 300 cells were analyzed for each data point. Error bars show the mean ± SD from three independent experiments (*P<0.05, **P<0.01). ATM, ataxia-telangiectasia mutated; IR, ionizing radiation.

Discussion

The standard radiotherapy for cervical cancer is the combination of external beam radiotherapy and intracavitary brachytherapy, which have the same efficiency on suppressing tumor growth and inhibiting metastasis as surgery for early patients (18). Radiation therapy serves as a major treatment strategy and is beneficial for cervical cancer patients to prevent recurrence or metastasis. Findings of a recent study showed that patients who received radiotherapy were 40–90% less likely to have a relapse of their cancer within 5 years than patients who did not undergo this therapy (3). However, we found a considerable number of patients suffering from radiation insensitivity in clinical practice and the 5-year survival rate was limited. Although there are constant technological innovations on radiotherapy equipment, its effect remains unsatisfactory, especially with respect to its negative effects of local recurrence and distant metastasis (19). Therefore, it is crucial to find predictive markers of radiation sensitivity.

The clinical value of miRNAs has been assessed because their expression is associated with tumor development, progression and even response to therapy (5,20,21). As previously reported, miRNAs were involved in the regulation of cellular radiosensitivity of various types of cancer. For instance, in breast cancer, miR-18a increased cellular radiosensitivity (13). miR-18a is located in the chromosome 13q31.1 region and belongs to the miR-17–92 cluster (22). A high expression of miR-18a has been found in several cancer types, such as bladder cancer (23), hepatocellular carcinoma (24), colon cancer (25) and nasopharyngeal carcinoma (26). The function of miR-18a in those types of cancer is as oncogene or suppressor. However, the functional role of miR-18a in cervical cancer remains unknown.

In the present study, we investigated the value of miR-18a in predicting the radiosensitivity of cervical cancer and reported, to the best of our knowledge, for the first time that the expression of miR-18a was significantly different between radiosensitive and radioresistant patients. The results showed that patients with a high expression of miR-18a were hypersensite to radiation as compared to patients with a low expression of miR-18a. The ROC curve indicated miR-18a may be a novel biomarker for predicting the radiosensitivity in cervical cancer treatment. In vitro, we demonstrated that overexpression of miR-18a significantly increases cellular sensitivity to radiotherapy using a colony formation assay, while the downregulation of miR-18a induced cell resistance to radiation. Furthermore, we revealed the molecular mechanism of how miR-18a altered the sensitivity of cancer cells to radiotherapy. miR-18a increased the radiosensitivity of cancer cells by targeting the 3′-UTR of the ATM gene, thereby reducing DSB repair. The most lethal form of DNA damage after radiation treatment is known to be the DSB. Cells suffering from DSB must be repaired for cells to survive, or it would lead to genomic instability and eventually apoptosis. DSB can be repaired by the two major pathways: non-homologous end joining (NHEJ) and homologous recombination repair (HRR) (27). DNA damage activates ATM through auto-phosphorylation and the activated ATM is thought to be the main regulator of HRR (16). DNA DSB can be induced after exposure to irradiation. ATM is then recruited to the DSB site and activated by auto-phosphorylation. Activated ATM can phosphorylate the histone variant H2AX on its C-terminal tail on serine 139, forming γ-H2AX foci. A sequence of downstream targets involving events to repair the DNA damage are activated, leading to amplification of the HRR signaling cascade (28,29). ATM has been demonstrated as a direct target of several miRNAs, and overexpression of those miRNAs downregulates ATM expression and sensitizes various cell lines to radiation therapy (30–32). It is known that miR-18a suppresses ATM expression by specifically targeting the 3′-UTR of ATM transcripts as previously reported. In this study, we upregulated and downregulated the expression of miR-18a in cervical cancer cells (SiHa and HeLa) by trans-fecting with miR-18a mimic and inhibitor. Tthe results showed that the level of miR-18a had no effect on the proliferation and apoptosis of the SiHa and HeLa cells post-transfection, but affected the expression of ATM after radiotherapy. Western blot analysis showed that overexpression of miR-18a resulted in a significantly decrease of ATM proteins and a sequential downregulation of the phosphorylation level of ATM downstream genes, such as γ-H2AX. Our data also suggest that overexpression of miR-18a increases cellular sensitivity to radiotherapy by enhancing radiation-associated apoptosis. To confirm the role of miR-18a in radiotherapy, we detected the expression level of caspase-3 and PARP. The results show that cleaved caspase-3 and PARP were markedly increased in miR-18a-upregulated cells than the controls, and vice versa.

In summary, we have demonstrated that the expression of miR-18a was significantly higher in the radiosensitive than in the radioresistant patients. ATM, a key protein to DNA damage repair, was the target of miR-18a in cervical cancer lines. In addition, overexpression of miR-18a suppressed the level of ATM and attenuated DNA DSB repair, which re-sensi-tized the cervical cancer cells to radiotherapy by promoting apoptosis. The results have demonstrated that the suppressed DNA damage repair induced by upregulated miR-18a may play an important role in radiation sensitivity. miR-18a may be a potential molecular marker for the detection of the response to radiotherapy in cervical cancer patients and a therapeutic target.

Acknowledgments

The authors thank the Gynecology and Obstetric Laboratory of Wuhan Union Hospital for providing the SiHa and HeLa cells, and Bohan Yang and Tianhe Zeng for their advice and assistance.

Abbreviations:

References