MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance by Targeting A20/NF-κB Signaling Pathway

Nasopharyngeal carcinoma (NPC) is the most frequent head and neck tumor in southern China and Southeast Asia, which poses one of the most serious public health problems in these areas (1). Radiotherapy is the major therapeutic modality used to treat NPC. Although NPC is sensitive to radiotherapy, a major impediment to achieve long-term survival is radioresistance that has been linked to an increased likelihood of recurrence and a distant metastasis (2, 3). To realize the full potential of radiotherapy, it is essential to understand the molecules and signaling pathways that mediate NPC radioresistance, a poorly characterized phenomenon, and identify druggable targets for radiosensitization.

MicroRNAs (miRNA), a class of endogenous noncoding RNAs, act as negative gene regulators at posttranscriptional level. MiRNAs are believed to play fundamental roles in the human cancers and have great potential in the diagnosis and treatment of cancers (4). Regulation of tumor radiosensitivity via miRNAs-associated mechanisms has attracted much attention in the recent years, and several miRNAs involving in tumor radioresistance have been identified (5–12). We previously used microarrays to compare the differences of miRNAs in the NPC cell lines with different radiosensitivity and found that miR-125b is one of upregulated miRNAs in the radioresistant NPC cells (NCBI-GSE48503). However, the function and mechanism of miR-125b in NPC radioresistance need to be elucidated.

NF-κB is an important regulator of cell proliferation and survival (13). Activation of the NF-κB signaling pathway not only plays a crucial role in the development and progression of NPC (14–16), but also confers tumor resistance to radiotherapy (17–20). Ubiquitin modification, occurring at multiple steps within the NF-κB signaling cascades, serves as a regulator in NF-κB activation (21). A growing number of proteins, such as receptor-interacting protein kinase 1 (RIP-1), TNF receptor–associated factor 2 (TRAF2), and TRAF6, in the NF-κB signal pathway have been identified to be modified by ubiquitin (21–24). Tumor necrosis factor alpha–induced protein 3 (TNFAIP3, also known as A20), functioning as an ubiquitin-editing enzyme (22), negatively regulates NF-κB signaling through dual mechanisms, i.e., deconjugation of K63-linked polyubiquitin chains from RIP-1 and subsequent conjugation of RIP-1 with K48-linked polyubiquitin chains for proteasomal degradation (23, 24). A20 can also catalyze the cleavage of K63-linked ubiquitin chains and the conjugation of K48-linked polyubiquitin chains, thereby targeting TRAF2 and TRAF6 for proteasomal degradation (25, 26). Therefore, A20 serves as a negative regulator in the NF-κB signal pathway by proteasomal degradation of its upstream signaling transducers.

Several studies have reported that A20 is a direct target of miR-125b, and miR-125b activates the NF-κB signaling pathway by inhibiting A20 expression (27–29). Numerous studies have demonstrated that A20 is involved in the pathogenesis of various types of human tumors. In some tumor types, A20 functions as a tumor suppressor due to its genetic or epigenetic inactivation, leading to A20 downregulation (30–32). In other tumors, A20 is upregulated and acts as oncogene (33–35). However, the roles of A20 in tumor radioresistance are unclear. Moreover, it is unknown whether NF-κB signaling mediates miR-125b/A20-regulating NPC radioresistance.

In the present study, we investigate whether and how miR-125b and A20 regulate NPC radioresistance. Here, we report that miR-125b is significantly upregulated, whereas A20 is significantly downregulated in the radioresistant NPCs relative to radiosensitive ones, and both are significantly correlated with poor patient survival; miR-125b increment confers NPC cell radioresistance both in vitro and in vivo by targeting the A20/NF-κB signaling pathway; A20 decrement also confers NPC cell radioresistance both in vitro and in vivo. These results can be extrapolated to clinical cases of NPC, as miR-125b level is significantly correlated with radioresistance and the levels of A20 and p-p65 (RelA) in NPC biopsies. Our findings demonstrate that both miR-125b and A20 are key molecules involved in NPC radioresistance, suggesting that NPC patients might benefit from radiosensitization therapies directed at miR-125b and A20.

One hundred and eleven NPC patients without distant metastasis (M0 stage) at the time of diagnosis who were treated by radical radiotherapy alone in the Affiliated Cancer Hospital of Central South University, China, between January 2006 and December 2008 were recruited in this study. The radiotherapy was administered for a total dose of 60 to 70 Gy (2 Gy/fraction, 5 days a week). The neck received 60 Gy for lymph node–negative cases and 70 Gy for lymph node–positive cases. NPC tissue biopsies were obtained at the time of diagnosis before any therapy, fixed in 4% formalin, and embedded in paraffin. We also acquired 30 cases of formalin-fixed and paraffin-embedded normal nasopharyngeal mucosa (NNM) in the same period. On the basis of the 1978 WHO classification (36), all tumors were histopathologically diagnosed as poorly differentiated squamous cell carcinomas (WHO type III). The clinical stage of the patients was classified according to the 2008 NPC staging system of China (37).

The radiotherapy response was evaluated clinically for primary lesions based on nasopharyngeal fiberscope and MRI 1 month after the initiation of radiotherapy according to the criteria as described previously by us (38). Based on the criteria, 111 NPC patients comprised 53 radioresistant and 58 radiosensitive ones.

The patients were followed up, and the follow-up period at the time of analysis was more than 72 months (average, 77.5 ± 11.8 months). Disease-free survival (DFS) was calculated as the time from the completion of primary radiotherapy to the date of pathologic diagnosis or clinical evidence of local failure and/or distant metastasis. Overall survival (OS) was defined as the time from the initiation of primary radiotherapy to the date of cancer-related death or when censured at the latest date if patients were still alive. The clinicopathologic parameters of the patients used in the present study are shown in Supplementary Table S1.

NPC cell line CNE-2 was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences in 2010 and was maintained in our laboratory. Radioresistant human NPC CNE2-IR cells and paired radiosensitive CNE2 cells were established previously by us (39) and cultured with RPMI-1640 medium containing 10% FBS (Life Technologies). Radioresistant CNE2-IR cells were derived from parental CNE2 cells by treating the cells with four rounds of sublethal ionizing radiation (39). Radiosensitive CNE2 cells, used as a control, were treated with the same procedure except sham irradiated. The cell lines were authenticated by DNA fingerprinting analysis using short-tandem repeat markers before the start of this study. Experiments were performed with the CNE2-IR cells within 4 to 10 passages after the termination of irradiation, and their radioresistance was tested by a clonogenic survival assay before use. The cell lines were routinely tested for the presence of mycoplasma with 4,6-diamidino-2-phenylindole staining.

Total RNA was extracted from cells with Trizol reagent (Life Technologies), or from the formalin-fixed and paraffin-embedded tissues with RecoverAll total nucleic acid isolation kit (Ambion) according to the manufacturer's instructions. For miR-125b qRT-PCR, 2 μg of total RNA was reversely transcribed for cDNA using a reverse transcription (RT) kit according to the manufacturer's instructions (Promega) and miR-125b–specific primer (Bulge-Loop miRNA qPCR primer). The RT products were amplified by real-time PCR using the miScript SYBR green PCR Kit (Qiagen) according to the manufacturer's instructions. For A20 mRNA qRT-PCR, 2 μg of total RNA was reversely transcribed for cDNA using an RT kit according to the manufacturer's protocol and Oligo dT primer (Promega) according to the manufacturer's instructions. The RT products were amplified by real-time PCR using a QuantiFast SYBR green PCR kit (Qiagen) according to the manufacturer's instructions. The products were quantitated using the 2^−DDCt method against U6 or GAPDH for normalization. The primer sequences were synthesized by RiboBio and summarized in Supplementary Table S2.

For the A20 3′UTR luciferase reporter assay, a dual-luciferase reporter plasmid with A20 3′UTR (GeneCopoeia), without A20 3′UTR (GeneCopoeia), or with mutated A20 3′UTR in the predicted miR-125b–binding site constructed by GeneCopoeia, and miR-125b or control mimic (RiboBio) was cotransfected into NPC cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For the NF-κB luciferase reporter assay, a dual-luciferase reporter plasmid containing human NF-κB/p65 response element (pNF-κB-TA-luc; Beyotime) or pGL6-TA plasmid without NF-κB/p65 response element (Beyotime) and pRL-TK plasmid (Promega) were cotransfected into NPC cells using Lipofectamine 2000. Cells were harvested 48 hours after transfection, both firefly luciferase and renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions, and luciferase activity was estimated using a luminometer (Promega).

Fifty and 100 μmol/L miR-125b mimic, miR-125b inhibitor, and their respective negative control (Ribobio) were transfected into cells using a RiboFect CP transfection kit (Ribobio) according to the manufacturer's instructions, respectively. Twenty-four hours after transfection, cells were subjected to further analysis.

Lentiviral GV248 vector expressing A20 shRNA or scramble nontarget shRNA, which was established by Genechem Co., and confirmed by sequencing. The target for human A20 shRNA was 5′-CACTGGAAGAAATACACATAT-3′, the knockdown (KD) efficiency of which has been validated (35). A20 expression plasmid (EX-K6040) and control plasmid pReceiver-M13 were purchased from GeneCopoeia. Cells were infected or transfected with the lentiviral vectors or plasmids according to the manufacturer's instructions, and then selected using neomycin or puromycin for 2 weeks. NPC cell lines with stable KD or overexpression (OE) of A20 and control cell lines were obtained.

A clonogenic survival assay was performed as previously described by us (38, 39). Briefly, cells were exposed to a range of radiation doses (0–10 Gy), and 12 days after irradiation, surviving colonies were stained with 0.5% crystal violet and counted. The survival fraction was calculated as the numbers of colonies divided by the numbers of cells seeded times plating efficiency. Radiation dose–response curves were created by fitting the data to the linear quadratic equation S = e^{−αD-βD⁁2} using GraphPad Prism 5.0, where S is the surviving fraction, α and β are inactivation constants, and D is the dose in Gy. The AUC that represents the mean inactivation dose (MID) was calculated using GraphPad Prism 5.0. The radiation protection factor was calculated by dividing the MID of the test cells by the MID of control cells.

Nude male mice that were 4 weeks old were obtained from the Laboratory Animal Center of Central South University and were maintained under specific pathogen-free conditions. For analyzing the effects of miR-125b on NPC radiosensitivity in vivo, 5 × 10⁶ CNE2-IR cells were subcutaneously injected into the right flanks of 5-week-old nude mice. When the xenograft volumes reached approximately 50 mm³, the transplanted mice were randomly divided into 2 groups (n = 5 mice each), 10 nmol control or miR-125b antagomir (RiboBio) in 25 μL saline buffer was intratumorally injected into the tumor mass at multiple sites per mouse, and next 3 days, a total dose of 6 Gy ionizing radiation (2 Gy/fraction, once per day) was delivered to the tumor. Three days after irradiation, 10 nmol control or miR-125b antagomir was intratumorally injected into the tumor mass. For analyzing the effects of A20 on NPC radiosensitivity in vivo, 5 × 10⁶ NPC cells with KD or OE of A20 and their control cells were subcutaneously injected into the right flanks of 5-week-old nude mice (n = 10 mice each), respectively. When the xenograft volumes reached approximately 50 mm³, a total dose of 6 Gy ionizing radiation (2 Gy/fraction, one per day) was delivered to the tumor.

Three weeks after irradiation, the mice were killed by cervical dislocation, and their tumors were excised, weighted, and embedded in paraffin for TUNEL and immunohistochemical staining. Tumor volume (in mm³) was measured by caliper measurements performed every 3 to 4 days and calculated by using the modified ellipse formula (volume = length × width²/2).

Cell apoptosis was assessed by the Annexin V–FITC apoptosis detection Kit I (Becton Dickinson Biosciences) according to the manufacturer's instructions. Briefly, 5 × 10⁵ cells were collected by centrifugation, resuspended in 500 μL binding buffer, and stained with 5 μL Annexin V conjugated with fluorescein isothiocyanate (FITC) and 5 μL propidium iodide at room temperature in the dark for 15 minutes, and then immediately analyzed by a FACSCalibur System. The relative proportion of Annexin V–positive cells was determined using the CellQuest Pro software and counted as the percentage of apoptotic cells. The assay was performed in triplicate for three times.

Proteins were exacted from cells. An equal amount of protein in each sample was subjected to SDS-PAGE separation, followed by blotting onto a PVDF membrane. After blocking, blots were incubated with anti-A20 (ab92324; Abcom), p-IKKα/β (#2078; CST), p-IκBα (#2859; CST), p-p65(RelA) (#3033; CST), IKKα (#2682; CST), IκBα (#4812; CST), or p65(RelA) antibody (#4764; CST) overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibody (#A24531 or #A24512; Life Technologies) for 2 hours at room temperature. The signal was visualized with an enhanced chemiluminescence detection reagent (Pierce). β-Actin was detected as a loading control.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was performed to detect apoptotic cells in the formalin-fixed and paraffin-embedded tissue sections of xenograft tumors with the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instruction. Quantitative evaluation of apoptotic cells was done by examining the sections in ten random microscopic fields and counting the number of TUNEL-positive cancer cells among 1,000 carcinoma cells under the light microscope. The apoptotic index was expressed as positive cells per 100 cancer cells.

Immunohistochemical staining was performed on formalin-fixed and paraffin-embedded tissue sections. Briefly, after antigen retrieval, tissue sections were incubated with anti-A20, p-p65(RelA), or γH2AX antibody (ab2893; Abcom) overnight at 4°C, and then were incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO). Finally, tissue sections were incubated with 3′, 3′-diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxylin. The immunoreactions were evaluated independently by two pathologists as described previously (38, 39). A staining score of ≤3 was considered to be low expression; and a score of >3 was considered to be high expression. Quantitative evaluation of DNA-damaged cells was done by examining the sections in ten random microscopic fields and counting the number of γH2AX-positive nuclei among 1,000 carcinoma cells under the light microscope. The rate of DNA-damaged cells was expressed as positive cells per 100 cancer cells.

Cells (2 × 10³) were plated into chamber slides (Millipore) and cultured with RPMI-1640 medium containing 10% FBS for 12 hours. Cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then cell membranes were permeabilized with 0.1% Triton 100 at room temperature for 20 minutes. Cells were washed with 1 × PBS and blocked with 10% goat serum in PBS for 1 hour. Then cells were incubated with p-p65 (RelA) overnight at 4°C. After washing with 1 × PBS for 3 times, cells were incubated with secondary antibodies conjugated with Alexa Fluor 594 (DI-1794; Vector Laboratories) for 1 hour. The slides were washed 3 times with 1 × PBS, counterstained with DAPI, mounted, and stored at 4°C under dark conditions. Pictures were taken under a Leica DMI4000 microscope.

All experiments were carried out at least 3 times. Data were presented as the mean ± SD. Statistical analysis was conducted using SPSS 22.0 statistical software package. For comparisons between two groups, a Student t test, χ² test, or Wilcoxon and Mann–Whitney test was used, and for analysis with multiple comparisons, Oneway ANOVA test was used. Survival curves were obtained by using the Kaplan–Meier method, and comparisons were made by using the log-rank test. Univariate and multivariate survival analyses were conducted on all parameters by using Cox proportional hazards regression model. The Spearman rank correlation coefficient was used to determine the correlation between two parameters. P values less than 0.05 were considered to be statistically significant.

This study was approved by the ethics committee of Xiangya School Medicine, Central South University, China. Written informed consent was obtained from all participants in the study. All animal experiments were undertaken in accordance with the Guide for the Care and Use of Laboratory Animals of Central South University, with the approval of the Scientific Investigation Board of Central South University.

Our previous microarray analysis found that miR-125b is upregulated in the radioresistant NPC CNE2-IR cells relative to paired radiosensitive CNE2 cells (NCBI-GSE48503). Using qRT-PCR, we confirmed that miR-125b expression was significantly increased in the CNE2-IR cells relative to CNE2 cells (Fig. 1A). We proceeded to detect miR-125b and A20 levels in 111 NPCs with the different radiosensitivity and 30 NNM by qRT-PCR and IHC, respectively. We observed that miR-125b was significantly increased, whereas A20 significantly decreased in the NPCs relative to NNM, and in the radioresistant NPCs relative to radiosensitive NPCs (Fig. 1A and B; Supplementary Table S3); miR-125b level was positively while A20 levels negatively correlated with NPC radioresistance (miR-125b; r = 0.72, P < 0.001; A20; r = −0.31, P < 0.01). Survival analyses revealed that both high miR-125b and low A20 levels in NPC tissues were correlated with the markedly reduced DFS and OS of the patients (Fig. 1C). A univariate and multivariate Cox regression analysis showed that both high miR-125b and low A20 expression were independent predictors for the reduced DFS and OS of NPC patients (Supplementary Table S4). These results indicate the importance of both miR-125b and A20 in the NPC radioresistance and prognosis.

To determine the effect of increased miR-125b on NPC cell radioresistance in vitro, CNE2-IR and CNE2 cells were transiently transfected with miR-125b inhibitor and mimic, respectively, and then cell radiosensitivity was determined. A clonogenic survival assay showed that miR-125b inhibitor significantly decreased while miR-125b mimic significantly increased NPC cell radioresistance compared with control inhibitor or mimic (Fig. 2A).

Irradiation primarily leads to double-strand DNA breaks (DSB), and unrepaired or misrepaired DSBs in the DNA lead to cell apoptosis. The apoptosis resulting from irradiation is, to a considerable degree, understood as radiosensitivity (40). Therefore, we analyzed the effect of miR-125b on the irradiation-induced apoptosis of NPC cells by using flow cytometry. The results showed that miR-125b inhibitor significantly increased while miR-125b mimic significantly decreased irradiation-induced apoptosis of NPC cells compared with control inhibitor or mimic 72 hours after 6 Gy irradiation (Fig. 2B). Taken together, these results demonstrate that miR-125b increases in vitro NPC cell radioresistance.

To determine the effect of miR-125b on NPC cell radioresponse in vivo, we generated subcutaneous tumors in nude mice using radioresistant CNE2-IR cells. Control or miR-125b antagomir was injected into the tumors before and after 6 Gy ionizing radiation, and then tumor radioresponse was assessed. As shown in Fig. 2C, radioresistance of miR-125b antagomir–injected tumors was significantly lower than that of control antagomir–injected tumors as demonstrated by tumor growth and weight. TUNEL assay showed that more apoptotic cells were present in the miR-125b antagomir–injected tumors relative to control antagomir–injected tumors (Fig. 2D). Immunohistochemical staining indicated that more positive cells of γH2AX, i.e., more cells with DNA damage, were present in the miR-125b antagomir–injected tumors compared with control agomir–injected tumors (Fig. 2D). Taken together, these results demonstrate that miR-125b increases in vivo NPC radioresistance and suggest that in vivo administration of miR-125b antagomir has a considerable potential for NPC radiosensitization.

To confirm A20 as a direct target of miR-125b, we cotransfected a dual-luciferase reporter plasmid with wild-type A20 3′UTR into CNE2 cells with control or miR-125b mimic. The results revealed a significant reduction in luciferase activity in miR-125b mimic–transfected cells compared with control mimic–transfected cells, whereas miR-125b mimic had no obvious effects on the luciferase activity of a dual-luciferase reporter plasmid without A20 3′UTR or with mutated A20 3′UTR in the miR-125b–binding site (Fig. 3A), confirming that miR-125b directly binds to A20 3′UTR Moreover, miR-125b mimic significantly decreased while miR-125b inhibitor significantly increased A20 levels in NPC cells compared with control mimic or inhibitor (Fig. 3A). Together, these results prove that A20 is a direct target of miR-125b in NPC cells.

Next, we analyzed whether A20 mediated miR-125b–regulated NPC cell radioresponse. CNE2-IR cell lines with A20 OE, CEN2 cell lines with A20 KD, and their control cell lines were established (Fig. 3B), and then cell radioresistance was detected by a clone survival assay and cell apoptosis analysis. The results showed that A20 OE significantly decreased, whereas A20 KD significantly increased NPC cell radioresistance (Fig. 3C and D), phenocopying those seen in the miR-125b inhibitor and mimic-transfected NPC cells, respectively. The result demonstrates that A20 decreases NPC cell radioresistance in vitro. Importantly, A20 KD markedly abolished the radiosensitizing effect of miR-125b inhibitor in the radioresistant CNE2-IR cells (Fig. 4A), and A20 OE markedly abolished radioresistance induced by miR-125b mimic in the radiosensitive CNE2 cells (Fig. 4B). Taken together, our results demonstrate that miR-125b regulates NPC cell radioresponse through targeting A20.

Our results showed that A20 regulated in vitro NPC cell radiosensitivity (Fig. 3B–D). To analyze the effects of A20 on NPC radiosensitivity in vivo, subcutaneous tumors of A20 KD CNE2 cells, A20 OE CNE2-IR cells, and their vector cells in nude mice received a total dose of 6 Gy ionization radiation, and then their radioresponse was monitored. The results showed that A20 KD significantly increased while A20 OE significantly decreased the radioresistance of xenograft tumors as demonstrated by tumor growth and weigh (Fig. 5A and B). TUNEL assay showed that A20 KD significantly decreased while A20 OE significantly increased the number of apoptotic cells in the xenograft tumors (Fig. 5C). Immunohistochemistry showed that A20 KD significantly decreased while A20 OE significantly increased the number of γH2AX-positive cells in the xenograft tumors (Fig. 5C). Moreover, A20 KD significantly increased while A20 OE significantly decreased the expression of p-p65 (RelA) in the xenograft tumors (Fig. 5C). Collectively, these results demonstrate that A20 decreases NPC cell radioresistance in vivo, supporting that miR-125b regulates NPC cells radiosensitivity in vivo through targeting A20.

Previous studies have revealed that activation of NF-κB confers tumor resistance to radiotherapy (17–20), and A20 is a negative regulator of the NF-κB signaling pathway (22–26). Therefore, we investigated whether NF-κB mediates miR-125b/A20-regulating NPC cell radioresponse. The results showed that either miR-125b mimic or A20 KD significantly enhanced the phosphorylated levels of IKKα/β, IκBα, and p65, p65 luciferase reporter activity, and the nuclear translocation of p-p65 in the radiosensitive CNE2 cells, whereas either miR-125b inhibitor or A20 OE significantly reduced the phosphorylated levels of IKKα/β, IκBα, and p65, p65 luciferase reporter activity, and the nuclear translocation of p-p65 in the radioresistant CNE2-IR cells (Fig. 6A–C). Importantly, A20 OE abrogated the effect of miR-125b inhibitor on NF-κB activity in the radiosensitive CNE2 cells, and A20 KD restored activity of NF-κB decreased by miR-125b mimic in the radioresistant CNE2-IR cells (Fig. 6D). Collectively, these results demonstrate that miR-125b regulates activity of the NF-κB signaling pathway by targeting A20.

Next, we determined whether NF-κB mediates miR-125b/A20-regulating NPC cell response. We observed that either IκBα OE or NF-κB inhibitor BAY11-7082 significantly abolished radioresistance induced by A20 KD in the radioresistant CNE2 cells, whereas NF-κB p65 OE restored radioresistance reduced by A20 OE in the radiosensitive CNE2-IR cells (Fig. 7A–C). These results demonstrate that NF-κB signaling mediates miR-125b/A20-regulating NPC cell radioresponse.

Because our data demonstrate that miR-125b regulates NPC cell radioresponse through targeting A20/NF-κB, we next determined whether the levels of miR-125b, A20, and p-p65 were correlated in NPC biopsies. Our IHC analysis showed that A20 expression was significantly lower while p-p65 was significantly higher in the radioresistant NPCs than that in the radiosensitive NPCs (Fig. 1C; Supplementary Table S3). Correlation analyses revealed that miR-125b level was negatively associated with A20 level (r = −0.61, P < 0.001), whereas positively associated with p-p65 level (r = 0.45, P < 0.001), and A20 level was negatively associated with p-p65 level (r = −0.38, P < 0.001). The results indicate that NF-κB signaling might mediate miR-125b/A20-regulating the radioresponse of clinical NPCs.

Radioresistance is the main obstacle in the clinical management of NPC (2, 3). Investigating the role of miRNAs in radioresistance is a promising avenue given their ability to regulate multiple oncogenic processes including response to therapy (41). In this study, we focused on miR-125b, one of upregulated miRNAs in the radioresistant NPC cells, because the function and mechanism of miR-125b in NPC radioresistance are unclear. We found that miR-125b increased NPC cell radioresistance both in vitro and in vivo. The finding is clinically relevant, given our discovery that miR-125b was frequently upregulated in radioresistant NPCs, and its increment was correlated with NPC radioresistance and poor patient survival, outlining a potential marker for predicting the radioresponse and prognosis of NPC patients, and suggesting its considerable potential in clinical NPC radiosensitization.

As miRNAs exert their roles through inhibiting target mRNA translation, thus identification of miR-125b target genes is a key step for understanding the mechanism of miR-125b–regulating NPC radioresistance. In this study, we confirm that A20 is a direct target of miR-125b in NPC cells, and miR-125b regulates NPC cell radioresponse through targeting A20. Numerous studies have revealed that A20, an ubiquitin-editing enzyme, negatively regulates activity of the NF-κB signaling pathway (22–26). Activation of the NF-κB signaling pathway not only plays a crucial role in the development and progression of NPC (14–16), but also confers tumor resistance to radiotherapy (17–20). Therefore, we investigated whether NF-κB mediates miR-125b/A20-regulating NPC cell radioresponse. We observed that miR-125b mimic or A20 KD enhanced, whereas miR-125b inhibitor or A20 OE reduced activity of NF-κB in NPC cells; A20 OE abrogated activity of NF-κB induced by miR-125b inhibitor, and A20 KD restored activity of NF-κB decreased by miR-125b mimic in the NPC cells, demonstrating that miR-125b activates NF-κB by targeting A20. We further showed that IκBα OE or BAY11-7082 could abolish radioresistance induced by A20 KD, whereas p65 (RelA) OE restored radioresistance reduced by A20 OE in the NPC cells. In the clinical NPC samples, p-p65 level was increased in the radioresistant NPCs relative to radiosensitive NPCs, and negatively associated with A20 level while positively associated with miR-125b level. Taken together, our results demonstrate that NF-κB mediates miR-125b/A20-regulating NPC cell radioresponse.

Numerous studies have indicated that A20 is involved in the pathogenesis of various types of human tumors (30–35). However, the roles of A20 in tumor radioresistance are unclear. In this study, we observed that A20 was decreased in the radioresistant NPCs relative to radiosensitive NPCs; A20 OE reduced while A20 KD enhanced NPC cell radioresistance in vitro and in vivo. The results strongly demonstrate that low A20 expression increases NPC radioresistance, suggesting its considerable potential in clinical NPC radiosensitization. To our knowledge, this is first reported that A20 regulates tumor radioresponse.

Although the A20/NF-κB signaling axis seems to largely account for the radioresistant phenotype of NPC cells induced by miR-125, indeed a single miRNA can target multiple mRNAs to regulate gene expression (41). Therefore, there might be other molecules such as BAK1, PPP1CA, and p53 (42–46), which are also targeted by miR-125b in NPC cells. We also observed that miR-125b negatively modulated p53 expression in NPC cells, but it still inhibited cell apoptosis in p53 KD NPC CNE2 cell line that was established previously by us (47). The results suggest that miR-125b increases NPC cell radioresistance by p53-independent manner, which is consistent with the previous report (45).

Methods for radiosensitization of NPC attract much attention (48–50). In this study, we observed that inhibition of miR-125b expression by using miR-125b antagomir enhanced NPC radiosensitivity in NPC xenografts. Nucleic-acid drugs, such as miRNAs, can be directly synthesized and modified to be more lipophilic that improves penetration. Such modification includes cholesterylation. Our delivery of miR-125b antagomir, cholesterylated miRNA inhibitor, successfully increased NPC radiosensitivity in intratumoral injection model, suggesting that miR-125b antagomir has a potential for further drug development.

In summary, our data demonstrate that miR-125b is frequently upregulated in the radioresistant NPCs and is an independent predictor for NPC survival; miR-125b regulates NPC cell radiosensitivity in vitro and in vivo through targeting A20 NF-κB signaling pathway; A20 is frequently downregulated in the radioresistant NPCs and is an independent predictor for NPC survival; A20 decreases NPC cell radioresistance both in vitro and in vivo. Our study demonstrates that both miR-125b and A20 are critical regulators of NPC radioresponse and suggests that targeting the miR-125b/A20/NF-κB signaling axis is a promising approach for enhancing NPC sensitivity to radiotherapy.

No potential conflicts of interest were disclosed.

Conception and design: L.-N. Li, T. Xiao, Z.-Q. Xiao

Development of methodology: L.-N. Li, T. Xiao

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L.-N. Li, T. Xiao, H.-M. Yi, Z. Zheng, J.-Q. Qu, X. Ye, H. Yi, S.-S. Lu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.-N. Li, T. Xiao, H.-M. Yi, J.-Q. Qu, W. Huang, X. Ye, X.-H. Li

Writing, review, and/or revision of the manuscript: T. Xiao, Z.-Q. Xiao

Study supervision: Z.-Q. Xiao

Other (processing tables and figures): W. Huang

We thank Dr. Tiebang Kang (The Cancer Center, Sun Yat-sen University, China) for providing pcDNA3.1-p65/RelA and pcDNA3.1-IκBα expression and control plasmids.

This work was supported by Major Program of the National Natural Science Foundation of China (81230053, to Z.-Q. Xiao), National Basic Research Program of China (2013CB910502, to Z.-Q. Xiao), and the National Natural Science Foundation of China (81472801, to X.-H. Li; 81672687, to Z.-Q. Xiao).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.