MiR-125b Increases Nasopharyngeal Carcinoma ......Small Molecule Therapeutics MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance by Targeting A20/NF-kB Signaling Pathway Li-Na

Small Molecule Therapeutics

MiR-125b Increases Nasopharyngeal CarcinomaRadioresistance by Targeting A20/NF-kBSignaling PathwayLi-Na Li1,2, Ta Xiao3, Hong-Mei Yi1, Zhen Zheng1, Jia-Quan Qu1,4,Wei Huang1,Xu Ye1, Hong Yi1, Shan-Shan Lu1, Xin-Hui Li1, and Zhi-Qiang Xiao1

Abstract

Radioresistance poses a major challenge in nasopharyngealcarcinoma (NPC) treatment, but little is known about howmiRNA regulates this phenomenon. In this study, we investigatedthe function and mechanism of miR-125b in NPC radioresis-tance, one of upregulated miRNAs in the radioresistant NPC cellsidentified by our previous microarray analysis. We observed thatmiR-125b was frequently upregulated in the radioresistant NPCs,and its increment was significantly correlated with NPC radio-resistance, and was an independent predictor for poor patientsurvival. In vitro radioresponse assays showed that miR-125binhibitor decreased, whereas miR-125b mimic increased NPCcell radioresistance. In a mouse model, therapeutic administra-tion of miR-125b antagomir dramatically sensitized NPC xeno-grafts to irradiation.Mechanistically, we confirmed that A20was adirect target of miR-125b and found that miR-125b regulated

NPC cell radioresponse by targeting A20/NF-kB signaling.With a combination of loss-of-function and gain-of-functionapproaches, we further showed that A20 overexpressiondecreased while A20 knockdown increased NPC cell radioresis-tance both in vitro and in vivo. Moreover, A20 was significantlydownregulated while p-p65 (RelA) significantly upregulated inthe radioresistant NPCs relative to radiosensitive NPCs, andmiR-125b expression level was negatively associated with A20expression level, whereas positively associated with p-p65 (RelA)level. Our data demonstrate that miR-125b and A20 are criticalregulators of NPC radioresponse, and high miR-125b expressionenhances NPC radioresistance through targeting A20 and thenactivating the NF-kB signaling pathway, highlighting the thera-peutic potential of the miR-125b/A20/NF-kB axis in clinical NPCradiosensitization. Mol Cancer Ther; 16(10); 2094–106. �2017 AACR.

IntroductionNasopharyngeal carcinoma (NPC) is the most frequent head

and neck tumor in southern China and Southeast Asia, whichposes one of the most serious public health problems in theseareas (1). Radiotherapy is the major therapeutic modality usedto treat NPC. Although NPC is sensitive to radiotherapy, amajor impediment to achieve long-term survival is radioresis-tance that has been linked to an increased likelihood of recur-rence and a distant metastasis (2, 3). To realize the full poten-tial of radiotherapy, it is essential to understand the moleculesand signaling pathways that mediate NPC radioresistance, a

poorly characterized phenomenon, and identify druggabletargets for radiosensitization.

MicroRNAs (miRNA), a class of endogenous noncoding RNAs,act as negative gene regulators at posttranscriptional level. MiR-NAs are believed to play fundamental roles in the human cancersand have great potential in the diagnosis and treatment of cancers(4). Regulation of tumor radiosensitivity via miRNAs-associatedmechanisms has attractedmuch attention in the recent years, andseveral miRNAs involving in tumor radioresistance have beenidentified (5–12). We previously used microarrays to comparethe differences of miRNAs in the NPC cell lines with differentradiosensitivity and found that miR-125b is one of upregulatedmiRNAs in the radioresistant NPC cells (NCBI-GSE48503). How-ever, the function and mechanism of miR-125b in NPC radio-resistance need to be elucidated.

NF-kB is an important regulator of cell proliferation andsurvival (13). Activation of the NF-kB signaling pathway not onlyplays 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 stepswithin the NF-kB signaling cascades, serves as a regulator inNF-kB activation (21). A growing number of proteins, such asreceptor-interacting protein kinase 1 (RIP-1), TNF receptor–asso-ciated factor 2 (TRAF2), and TRAF6, in the NF-kB signal pathwayhave been identified to bemodified by ubiquitin (21–24). Tumornecrosis factor alpha–induced protein 3 (TNFAIP3, also known asA20), functioning as an ubiquitin-editing enzyme (22), negative-ly regulates NF-kB signaling through dual mechanisms, i.e.,

1Research Center of Carcinogenesis and Targeted Therapy, Xiangya Hospital,Central South University, Changsha, Hunan, China. 2Department of Pathology,Changzhi Medical College, Changzhi, Shanxi, China. 3Department of Dermatol-ogy, Xiangya Hospital, Central South University, Changsha, Hunan, China.4Department of Oncology, Qianjiang Central Hospital of Chongqing, JishouUniversity, Hunan, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

L.-N. Li, T. Xiao, and H.-M. Yi contributed equally to this article.

Corresponding Author: Z.-Q. Xiao, Xiangya Hospital, Central South University,87# Xiangya Road, Changsha, Hunan 410008, China. Phone: 86-731-89753378;Fax: 86-731-84327332; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0385

�2017 American Association for Cancer Research.

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deconjugation of K63-linked polyubiquitin chains from RIP-1and subsequent conjugation of RIP-1 with K48-linked polyubi-quitin chains for proteasomal degradation (23, 24). A20 can alsocatalyze the cleavage of K63-linked ubiquitin chains and theconjugation of K48-linked polyubiquitin chains, thereby target-ing TRAF2 and TRAF6 for proteasomal degradation (25, 26).Therefore, A20 serves as a negative regulator in the NF-kB signalpathway by proteasomal degradation of its upstream signalingtransducers.

Several studies have reported that A20 is a direct target ofmiR-125b, and miR-125b activates the NF-kB signaling pathwayby inhibiting A20 expression (27–29). Numerous studies havedemonstrated that A20 is involved in the pathogenesis of varioustypes of human tumors. In some tumor types, A20 functions as atumor suppressor due to its genetic or epigenetic inactivation,leading to A20 downregulation (30–32). In other tumors, A20 isupregulated and acts as oncogene (33–35). However, the roles ofA20 in tumor radioresistance are unclear.Moreover, it is unknownwhether NF-kB signaling mediates miR-125b/A20-regulatingNPC radioresistance.

In the present study,we investigatewhether andhowmiR-125band A20 regulate NPC radioresistance. Here, we report thatmiR-125b is significantly upregulated, whereas A20 is significant-ly downregulated in the radioresistant NPCs relative to radiosen-sitive ones, and both are significantly correlated with poor patientsurvival; miR-125b increment confers NPC cell radioresistanceboth in vitro and in vivo by targeting the A20/NF-kB signalingpathway; A20 decrement also confers NPC cell radioresistanceboth in vitro and in vivo. These results can be extrapolated toclinical cases of NPC, as miR-125b level is significantly correlatedwith radioresistance and the levels of A20 and p-p65 (RelA) inNPC biopsies. Our findings demonstrate that bothmiR-125b andA20 are key molecules involved in NPC radioresistance, suggest-ing that NPC patients might benefit from radiosensitizationtherapies directed at miR-125b and A20.

Materials and MethodsPatients and tissue samples

One hundred and eleven NPC patients without distant metas-tasis (M0 stage) at the time of diagnosis who were treated byradical radiotherapy alone in the Affiliated Cancer Hospital ofCentral South University, China, between January 2006 andDecember 2008 were recruited in this study. The radiotherapywas 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–negativecases and 70 Gy for lymph node–positive cases. NPC tissuebiopsies were obtained at the time of diagnosis before anytherapy, fixed in 4% formalin, and embedded in paraffin. Wealso acquired 30 cases of formalin-fixed and paraffin-embeddednormal nasopharyngeal mucosa (NNM) in the same period. Onthe basis of the 1978 WHO classification (36), all tumors werehistopathologically diagnosed as poorly differentiated squamouscell carcinomas (WHO type III). The clinical stage of the patientswas classified according to the 2008 NPC staging system ofChina (37).

The radiotherapy response was evaluated clinically for primarylesions based on nasopharyngeal fiberscope and MRI 1 monthafter the initiation of radiotherapy according to the criteria asdescribed previously by us (38). Based on the criteria, 111 NPCpatients comprised 53 radioresistant and 58 radiosensitive ones.

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

Cell linesNPC cell line CNE-2 was purchased from the Cell Bank of Type

Culture Collection of the Chinese Academy of Sciences in 2010and was maintained in our laboratory. Radioresistant humanNPC CNE2-IR cells and paired radiosensitive CNE2 cells wereestablished previously by us (39) and cultured with RPMI-1640medium containing 10% FBS (Life Technologies). RadioresistantCNE2-IR cells were derived from parental CNE2 cells by treatingthe cells with four rounds of sublethal ionizing radiation (39).Radiosensitive CNE2 cells, used as a control, were treated with thesame procedure except sham irradiated. The cell lines wereauthenticated byDNA fingerprinting analysis using short-tandemrepeat markers before the start of this study. Experiments wereperformedwith theCNE2-IR cells within 4 to 10 passages after thetermination of irradiation, and their radioresistance was tested bya clonogenic survival assay beforeuse. The cell lineswere routinelytested for the presence of mycoplasma with 4,6-diamidino-2-phenylindole staining.

QRT-PCRTotal RNA was extracted from cells with Trizol reagent (Life

Technologies), or from the formalin-fixed and paraffin-embed-ded tissues with RecoverAll total nucleic acid isolation kit(Ambion) according to the manufacturer's instructions. FormiR-125b qRT-PCR, 2 mg of total RNA was reversely transcribedfor cDNA using a reverse transcription (RT) kit according to themanufacturer's instructions (Promega) and miR-125b–specificprimer (Bulge-LoopmiRNA qPCR primer). The RT products wereamplified by real-time PCRusing themiScript SYBR green PCRKit(Qiagen) according to the manufacturer's instructions. For A20mRNA qRT-PCR, 2 mg of total RNA was reversely transcribed forcDNA using an RT kit according to the manufacturer's protocoland Oligo dT primer (Promega) according to the manufacturer'sinstructions. The RT products were amplified by real-time PCRusing a QuantiFast SYBR green PCR kit (Qiagen) according to themanufacturer's instructions. The products were quantitated usingthe 2�DDCt method against U6 or GAPDH for normalization. Theprimer sequences were synthesized by RiboBio and summarizedin Supplementary Table S2.

Luciferase activity assayFor the A20 30UTR luciferase reporter assay, a dual-luciferase

reporter plasmid with A20 30UTR (GeneCopoeia), without A2030UTR (GeneCopoeia), or with mutated A20 30UTR in the pre-dicted miR-125b–binding site constructed by GeneCopoeia, andmiR-125b or control mimic (RiboBio) was cotransfected intoNPC cells using Lipofectamine 2000 (Invitrogen) according to themanufacturer's instructions. For the NF-kB luciferase reporterassay, a dual-luciferase reporter plasmid containing human

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NF-kB/p65 response element (pNF-kB-TA-luc; Beyotime) orpGL6-TA plasmid without NF-kB/p65 response element (Beyo-time) and pRL-TK plasmid (Promega) were cotransfected intoNPC cells using Lipofectamine 2000. Cells were harvested48 hours after transfection, both firefly luciferase and renillaluciferase activities were measured using the dual-luciferasereporter assay system (Promega) according to the manufacturer'sinstructions, and luciferase activity was estimated using a lumin-ometer (Promega).

Transfection of miR-125b mimic and inhibitor into cellsFifty and 100 mmol/L miR-125b mimic, miR-125b inhibitor,

and their respective negative control (Ribobio) were transfectedinto cells using a RiboFect CP transfection kit (Ribobio) accordingto the manufacturer's instructions, respectively. Twenty-fourhours after transfection, cells were subjected to further analysis.

Establishment of NPC cell lines with A20 overexpression andknockdown

Lentiviral GV248 vector expressing A20 shRNA or scramblenontarget shRNA, which was established by Genechem Co., andconfirmed by sequencing. The target for human A20 shRNA was50-CACTGGAAGAAATACACATAT-30, the knockdown (KD) effi-ciency of which has been validated (35). A20 expression plasmid(EX-K6040) and control plasmid pReceiver-M13 were purchasedfrom GeneCopoeia. Cells were infected or transfected with thelentiviral vectors or plasmids according to the manufacturer'sinstructions, and then selected using neomycin or puromycin for2 weeks. NPC cell lines with stable KD or overexpression (OE) ofA20 and control cell lines were obtained.

Clonogenic survival assayA clonogenic survival assay was performed as previously

described by us (38, 39). Briefly, cells were exposed to a rangeof radiation doses (0–10 Gy), and 12 days after irradiation,surviving colonies were stained with 0.5% crystal violet andcounted. The survival fraction was calculated as the numbers ofcolonies divided by the numbers of cells seeded times platingefficiency. Radiation dose–response curves were created by fittingthe data to the linear quadratic equation S ¼ e�aD-bD^2 usingGraphPad Prism 5.0, where S is the surviving fraction, a and b

are inactivation constants, and D is the dose in Gy. The AUCthat represents the mean inactivation dose (MID) was calculatedusing GraphPad Prism 5.0. The radiation protection factor wascalculated by dividing the MID of the test cells by the MID ofcontrol cells.

In vivo tumor radioresponse assayNude male mice that were 4 weeks old were obtained from the

Laboratory Animal Center of Central South University and weremaintained under specific pathogen-free conditions. For analyz-ing the effects ofmiR-125bonNPC radiosensitivity in vivo, 5�106

CNE2-IR cells were subcutaneously injected into the right flanksof 5-week-old nude mice. When the xenograft volumes reachedapproximately 50 mm3, the transplanted mice were randomlydivided into 2 groups (n ¼ 5 mice each), 10 nmol control ormiR-125b antagomir (RiboBio) in 25 mL saline buffer was intra-tumorally injected into the tumor mass at multiple sites permouse, and next 3 days, a total dose of 6 Gy ionizing radiation(2 Gy/fraction, once per day) was delivered to the tumor. Threedays after irradiation, 10 nmol control or miR-125b antagomir

was intratumorally injected into the tumor mass. For analyzingthe effects of A20 on NPC radiosensitivity in vivo, 5 � 106 NPCcells with KD or OE of A20 and their control cells were subcu-taneously injected into the rightflanksof 5-week-oldnudemice (n¼ 10 mice each), respectively. When the xenograft volumesreached approximately 50 mm3, a total dose of 6 Gy ionizingradiation (2 Gy/fraction, one per day) was delivered to the tumor.

Three weeks after irradiation, the mice were killed by cervicaldislocation, and their tumors were excised, weighted, and embed-ded in paraffin for TUNEL and immunohistochemical staining.Tumor volume (in mm3) was measured by caliper measurementsperformed every 3 to 4 days and calculated by using the modifiedellipse formula (volume ¼ length � width2/2).

Flow cytometry analysisCell apoptosis was assessed by the Annexin V–FITC apoptosis

detection Kit I (Becton Dickinson Biosciences) according to themanufacturer's instructions. Briefly, 5�105 cellswere collectedbycentrifugation, resuspended in 500 mL binding buffer, and stainedwith 5 mL Annexin V conjugated with fluorescein isothiocyanate(FITC) and 5 mL propidium iodide at room temperature inthe dark for 15 minutes, and then immediately analyzed by aFACSCalibur System. The relative proportion of Annexin V–positive cells was determined using the CellQuest Pro softwareand counted as the percentage of apoptotic cells. The assay wasperformed in triplicate for three times.

Western blottingProteinswere exacted from cells. An equal amount of protein in

each sample was subjected to SDS-PAGE separation, followed byblotting onto a PVDF membrane. After blocking, blots wereincubated with anti-A20 (ab92324; Abcom), p-IKKa/b (#2078;CST), p-IkBa (#2859; CST), p-p65(RelA) (#3033; CST), IKKa(#2682;CST), IkBa (#4812;CST), or p65(RelA) antibody (#4764;CST) overnight at 4�C, followed by incubation with horseradishperoxidase–conjugated secondary antibody (#A24531 or#A24512; Life Technologies) for 2 hours at room temperature.The signal was visualized with an enhanced chemiluminescencedetection reagent (Pierce). b-Actin was detected as a loadingcontrol.

In situ detection of apoptotic cellsTerminal deoxynucleotidyl transferase (TdT)-mediated dUTP

nick end labeling (TUNEL) was performed to detect apoptoticcells in the formalin-fixed and paraffin-embedded tissue sectionsof xenograft tumors with the In Situ Cell Death Detection Kit(Roche) according to themanufacturer's instruction.Quantitativeevaluation of apoptotic cells was done by examining the sectionsin ten random microscopic fields and counting the number ofTUNEL-positive cancer cells among 1,000 carcinoma cells underthe light microscope. The apoptotic index was expressed aspositive cells per 100 cancer cells.

ImmunohistochemistryImmunohistochemical staining was performed on formalin-

fixed and paraffin-embedded tissue sections. Briefly, after antigenretrieval, tissue sections were incubated with anti-A20, p-p65(RelA), or gH2AX antibody (ab2893; Abcom) overnight at 4�C,and then were incubated with biotinylated secondary antibodyfollowed by avidin-biotin peroxidase complex (DAKO). Finally,tissue sections were incubated with 30, 30-diaminobenzidine

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(Sigma-Aldrich) and counterstained with hematoxylin. Theimmunoreactions were evaluated independently by two pathol-ogists as described previously (38, 39). A staining score of�3 wasconsidered to be low expression; and a score of >3was consideredto be high expression. Quantitative evaluation of DNA-damagedcells was done by examining the sections in ten random micro-scopic fields and counting the number of gH2AX-positive nucleiamong 1,000 carcinoma cells under the lightmicroscope. The rateof DNA-damaged cells was expressed as positive cells per 100cancer cells.

Immunofluorescent stainingCells (2� 103)were plated into chamber slides (Millipore) and

cultured with RPMI-1640 medium containing 10% FBS for12 hours. Cells were fixed with 4% paraformaldehyde at roomtemperature for 15 minutes, and then cell membranes werepermeabilized with 0.1% Triton 100 at room temperature for20 minutes. Cells were washed with 1 � PBS and blocked with10% goat serum in PBS for 1 hour. Then cells were incubated withp-p65 (RelA) overnight at 4�C. After washing with 1 � PBS for3 times, cells were incubated with secondary antibodies conju-gated with Alexa Fluor 594 (DI-1794; Vector Laboratories) for1 hour. The slides were washed 3 times with 1 � PBS, counter-stained with DAPI, mounted, and stored at 4�C under darkconditions. Pictures were taken under a Leica DMI4000microscope.

Statistical analysisAll experiments were carried out at least 3 times. Data were

presented as the mean � SD. Statistical analysis was conductedusing SPSS 22.0 statistical software package. For comparisonsbetween two groups, a Student t test, c2 test, or Wilcoxon and

Mann–Whitney test was used, and for analysis with multiplecomparisons, Oneway ANOVA test was used. Survival curves wereobtained by using the Kaplan–Meier method, and comparisonswere made by using the log-rank test. Univariate andmultivariatesurvival analyses were conducted on all parameters by using Coxproportional hazards regression model. The Spearman rank cor-relation coefficientwas used to determine the correlation betweentwo parameters. P values less than 0.05 were considered to bestatistically significant.

Ethics statementThis study was approved by the ethics committee of Xiangya

School Medicine, Central South University, China. Writteninformed consent was obtained from all participants in the study.All animal experiments were undertaken in accordance with theGuide for the Care and Use of Laboratory Animals of CentralSouth University, with the approval of the Scientific InvestigationBoard of Central South University.

ResultsExpression of miR-125b and A20 is correlated with NPCradiosensitivity and patient prognosis

Our previous microarray analysis found that miR-125b isupregulated in the radioresistant NPC CNE2-IR cells relativeto paired radiosensitive CNE2 cells (NCBI-GSE48503). UsingqRT-PCR, we confirmed that miR-125b expression was signifi-cantly increased in the CNE2-IR cells relative to CNE2 cells (Fig.1A). We proceeded to detect miR-125b and A20 levels in 111NPCswith the different radiosensitivity and 30NNMby qRT-PCRand IHC, respectively. We observed that miR-125b was signifi-cantly increased, whereas A20 significantly decreased in the NPCsrelative to NNM, and in the radioresistant NPCs relative to

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Figure 1.

Association of miR-125b and A20 expression levels with NPC radioresistance and patients survival. A, qRT-PCR showing the expression levels of miR-125b inradioresistant CNE2-IR and paired radiosensitive CNE2 cells (left), NNM, and radiosensitive and radioresistant NPC tissues (right). Three experimentswere done; mean, SDs, and statistical significance are denoted; �� , P < 0.01; ��, P < 0.001. B, A representative result of immunohistochemistry showing theexpression levels of A20 and p-p65 (RelA) in NNM and radiosensitive and radioresistant NPC tissues. Original magnification, �400. C, Kaplan–Meier survivalanalysis for NPC patients according to the expression levels of miR-125b (top) or A20 (bottom). NPC patients with high miR-125b or low A20 expression havea poor DFS and OS. The log-rank test was used to calculate P value.



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radiosensitive NPCs (Fig. 1A and B; Supplementary Table S3);miR-125b level was positively while A20 levels negatively corre-lated with NPC radioresistance (miR-125b; r ¼ 0.72, P < 0.001;A20; r ¼ �0.31, P < 0.01). Survival analyses revealed that bothhighmiR-125b and low A20 levels in NPC tissues were correlatedwith themarkedly reducedDFS andOSof the patients (Fig. 1C). Aunivariate and multivariate Cox regression analysis showed thatboth high miR-125b and low A20 expression were independentpredictors for the reduced DFS and OS of NPC patients (Supple-mentary Table S4). These results indicate the importance of bothmiR-125b and A20 in the NPC radioresistance and prognosis.

MiR-125b increases NPC cell radioresistance in vitroTo determine the effect of increased miR-125b on NPC cell

radioresistance in vitro, CNE2-IR and CNE2 cells were transientlytransfected withmiR-125b inhibitor andmimic, respectively, andthen cell radiosensitivity was determined. A clonogenic survivalassay showed that miR-125b inhibitor significantly decreasedwhile miR-125b mimic significantly increased NPC cell radio-resistance 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 tocell apoptosis. The apoptosis resulting from irradiation is, to aconsiderable degree, understood as radiosensitivity (40). There-fore, we analyzed the effect of miR-125b on the irradiation-induced apoptosis of NPC cells by using flow cytometry. Theresults showed that miR-125b inhibitor significantly increasedwhile miR-125b mimic significantly decreased irradiation-induced apoptosis of NPC cells compared with control inhibitorormimic 72 hours after 6Gy irradiation (Fig. 2B). Taken together,these results demonstrate thatmiR-125b increases in vitroNPCcellradioresistance.

MiR-125b increases NPC cell radioresistance in vivoTo determine the effect ofmiR-125b onNPC cell radioresponse

in vivo, we generated subcutaneous tumors in nude mice usingradioresistant CNE2-IR cells. Control ormiR-125b antagomir wasinjected 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 wassignificantly lower than that of control antagomir–injectedtumors as demonstrated by tumor growth and weight. TUNELassay showed that more apoptotic cells were present in the miR-125b antagomir–injected tumors relative to control antagomir–injected tumors (Fig. 2D). Immunohistochemical staining indi-cated that more positive cells of gH2AX, i.e., more cells with DNAdamage, were present in the miR-125b antagomir–injectedtumors compared with control agomir–injected tumors(Fig. 2D). Taken together, these results demonstrate that miR-125b increases in vivoNPC radioresistance and suggest that in vivoadministration ofmiR-125b antagomir has a considerable poten-tial for NPC radiosensitization.

MiR-125b increases NPC cell radioresistance through targetingA20

To confirm A20 as a direct target ofmiR-125b, we cotransfecteda dual-luciferase reporter plasmid with wild-type A20 30UTR intoCNE2 cellswith control ormiR-125bmimic. The results revealed asignificant reduction in luciferase activity in miR-125b mimic–transfected cells compared with control mimic–transfected cells,whereasmiR-125bmimic had no obvious effects on the luciferase

activity of a dual-luciferase reporter plasmid without A20 30UTRor with mutated A20 30UTR in the miR-125b–binding site(Fig. 3A), confirming that miR-125b directly binds to A20 30UTRMoreover, miR-125b mimic significantly decreased while miR-125b inhibitor significantly increased A20 levels in NPC cellscompared with control mimic or inhibitor (Fig. 3A). Together,these results prove that A20 is a direct target of miR-125b inNPC cells.

Next, we analyzed whether A20mediatedmiR-125b–regulatedNPC cell radioresponse. CNE2-IR cell lines with A20 OE, CEN2cell lineswithA20KD, and their control cell lineswere established(Fig. 3B), and then cell radioresistance was detected by a clonesurvival assay and cell apoptosis analysis. The results showed thatA20 OE significantly decreased, whereas A20 KD significantlyincreasedNPC cell radioresistance (Fig. 3C andD), phenocopyingthose seen in themiR-125b inhibitor andmimic-transfected NPCcells, respectively. The result demonstrates that A20 decreasesNPC cell radioresistance in vitro. Importantly, A20 KD markedlyabolished the radiosensitizing effect of miR-125b inhibitor in theradioresistant CNE2-IR cells (Fig. 4A), and A20 OE markedlyabolished radioresistance induced by miR-125b mimic in theradiosensitive CNE2 cells (Fig. 4B). Taken together, our resultsdemonstrate that miR-125b regulates NPC cell radioresponsethrough targeting A20.

A20 decreases NPC cell radioresistance in vivoOur results showed that A20 regulated in vitro NPC cell radio-

sensitivity (Fig. 3B–D). To analyze the effects of A20 on NPCradiosensitivity in vivo, subcutaneous tumors of A20 KD CNE2cells, A20 OE CNE2-IR cells, and their vector cells in nude micereceived a total dose of 6 Gy ionization radiation, and then theirradioresponse was monitored. The results showed that A20 KDsignificantly increased while A20 OE significantly decreased theradioresistance of xenograft tumors as demonstrated by tumorgrowth and weigh (Fig. 5A and B). TUNEL assay showed that A20KD significantly decreased while A20 OE significantly increasedthe number of apoptotic cells in the xenograft tumors (Fig. 5C).Immunohistochemistry showed that A20 KD significantlydecreased while A20 OE significantly increased the number ofgH2AX-positive cells in the xenograft tumors (Fig. 5C).Moreover,A20 KD significantly increased while A20 OE significantlydecreased the expression of p-p65 (RelA) in the xenografttumors (Fig. 5C). Collectively, these results demonstrate thatA20 decreases NPC cell radioresistance in vivo, supporting thatmiR-125b regulates NPC cells radiosensitivity in vivo throughtargeting A20.

NF-kB mediates miR-125b/A20-regulating NPC cellradioresponse

Previous studies have revealed that activation of NF-kB conferstumor resistance to radiotherapy (17–20), and A20 is a negativeregulator of the NF-kB signaling pathway (22–26). Therefore, weinvestigated whether NF-kB mediates miR-125b/A20-regulatingNPC cell radioresponse. The results showed that either miR-125bmimic or A20 KD significantly enhanced the phosphorylatedlevels of IKKa/b, IkBa, and p65, p65 luciferase reporter activity,and the nuclear translocation of p-p65 in the radiosensitive CNE2cells, whereas either miR-125b inhibitor or A20 OE significantlyreduced the phosphorylated levels of IKKa/b, IkBa, and p65, p65luciferase reporter activity, and the nuclear translocation of p-p65in the radioresistant CNE2-IR cells (Fig. 6A–C). Importantly, A20

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Dose (Gy)

P < 0.05

P < 0.05

CNE2-IR+inhibitor controlCNE2-IR+miRNA-125b inhibitor (50 nmol/L)CNE2-IR+miRNA-125b inhibitor (100 nmol/L)

CNE2+mimic controlCNE2+miRNA-125b mimic (50 nmol/L)CNE2+miRNA-125b mimic (100 nmol/L)

RPF(50 nmol/L miRNA-125b inhibitor) = 0.77RPF(100 nmol/L miRNA-125b inhibitor) = 0.54

RPF(50 nmol/L miRNA-125b mimic) = 1.30


CNE2-IR

CNE2

Inhibitor control

Mimic control

miR-125b inhibitor

miR-125b mimic

50 nmol/L

50 nmol/L

100 nmol/L

100 nmol/L

35

30

25

20

15

10

5

0

Apo

ptot

ic ra

te (%

)

CNE2-IR CNE2

Inhi

bito

r con

trol

miR

-125

b in

hibi

tor

50 n

mol

/Lm

iR-1

25b

inhi

bito

r

100

nmol

/L

miR

-125

b m

imic

50 n

mol

/L

Mim

ic co

ntro

l

miR

-125

b m

imic

100

nmol

/L

2,000

1,500

1,000

500

00 3 6 9 12 15 18 21

Time after irradiation (day)

Tum

or v

olum

e (m

m3 )

Tum

or w

eigh

t (g)

CNE2-IR/control antagomir

CNE2-IR/control antagomir

CNE2-IR/miR-125b antagomir

CNE2-IR/miR-125b antagomir1.0

0.8

0.6

0.4

0.2

0.0

CNE2-IR

CNE2-IRControl antagomir miR-125b antagomir

Control antagomirmiR125b antagomir

A20

TUNEL

γH2AX

5

4

3

2

1

040

30

20

10

0

40

30

20

10

0

A20

Exp

ress

ion

scor

eP

erce

ntag

e of

apop

totic

cel

lsP

erce

ntag

e of

posi

tive

γH2A

X c

ells

A

B C

D

Figure 2.

MiR-125b increases NPC cell radioresistance in vitro and in vivo. A, A clonogenic survival assay showing the radioresponse of miR-125b inhibitor–transfectedCNE2-IR cells, miR-125b mimic–transfected CNE2 cells, and their control cells. Left, cells were irradiated with a range of 0 to 10 Gy radiation doses, and colonies thatformed after incubation of 12 days were stained with crystal violet and photographed; right, dose survival curves were created by fitting surviving fractionsto the linear quadratic equation.B, Flow cytometric analysis showing cell apoptosis in themiR-125b inhibitor–transfected CNE2-IR cells, miR-125bmimic–transfectedCNE2 cells, and their control cells 72 hours after 6 Gy ionizing radiation. Three experiments were done; mean, SDs, and statistical significance are denoted;�� , P < 0.01; �� , P < 0.001. C, miR-125b antagomir decreases in vivo NPC cell radioresistance. The gross appearance (left) and growth and weight (right) ofcontrol and miR-125b antagomir–injected CNE2-IR cell xenograft tumors (n ¼ 5 each group) 21 days after 6 Gy irradiation. D, Immunohistochemistry showingthe expression of A20 and gH2AX and TUNEL-detected apoptotic cells in control and miR-125b antagomir–injected CNE2-IR cell xenograft tumors 21 daysafter 6 Gy irradiation. Original magnification, �400. Mean, SDs, and statistical significance are denoted; �� , P < 0.01; �� , P < 0.001.



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Binding site: (496/503-8mer)120

100

80

60

40

20

0

Mimic controlmiR-125b mimic

Rel

ativ

e lu

cife

rase

act

ivity

(%)

A20 3'

-UTR W

T

A20 3'

-UTR m

ut1A20

3'-U

TR mut2

Vector

with

out

A20 3'

-UTR

A20 3'UTR

A20 3'UTR mutant1

A20 3'UTR mutant2

miR-125b 1995

496

496

503

503

CNE2 CNE2-IRCNE2-IR CNE2

Blank

Blank

Blank

Vector

A20-O

E1A20

-OE2

A20-K

D2

A20-K

D1

scr-s

hRNA

BlankMim

icco

ntrol

Inhibi

torco

ntrol

miR-12

5b

inhibi

tor

miR-12

5bmim

ic

1.00 1.16 0.73 1.00 1.32 2.77 1.0 1.0 0.78 0.30 0.201.07 1.67 1.71

A20

Tubulin Tubulin

A20

CNE2-IR

CNE2

CNE2-IR

CNE2



A20

-OE

2A

20-O

E1

Vect

orA

20-K

D2

A20

-KD

1sc

r-sh

RN

A

CNE2-IR

CNE2

CNE2-IR CNE2

0.1

0.01

0.001

0.1

0.01

0.001

0 2 4 6 8 10

0 2 4 6 8 10

Dose (Gy)

Sur

viva

l fra

ctio

nS

urvi

val f

ract

ion

VectorA20-OE1

A20-KD1A20-KD2

scr-shRNA

A20-OE2P < 0.05

P < 0.05

RPF(A20-OE1) = 0.59

RPF(A20-OE2) = 0.66

RPF(A20-KD1) = 1.55

RPF(A20-KD2) = 1.68

35

30

25

20

15

10

5

0

Apo

ptot

ic ra

te (%

)

Dose (Gy)

Vector

A20 O

E1A20

OE2

A20-K

D1A20

-KD2

scr-s

hRNA

Vector A20-OE1 A20-OE2

scr-shRNA A20-KD1 A20-KD2

A

C

D

B

Figure 3.

Target A20 of miR-125b regulates NPC cell radioresponse. A, A20 is a direct target of miR-125b. Left, the predicted miR-125b–binding sites in thewild-type (wt) A20 30UTR and mutant (mt) A20 30UTR are shown; right, luciferase activity of a dual-luciferase reporter vector with wt A20 30UTR, mt A20 30UTR,or without A20 30UTR in the CNE2 cells transfected with control or miR-125b mimic is shown; left bottom, Western blot analysis showing A20 levels inthe miR-125b mimic–transfected CNE2, inhibitor–transfected CNE2-IR cells, and their respective control cells. B, Western blot analysis showing A20 levels inthe A20 OE CNE2-IR cells, A20 KD CNE2 cells, and their respective control cells. C, A clonogenic survival assay showing the radioresponse of A20 OE CNE2-IR cells,A20 KD CNE2 cells, and their respective control cells. Left, Cells were irradiated with a range of 0 to 10 Gy radiation doses, and colonies that formed afterincubation of 12 dayswere stained with crystal violet and photographed; right, dose survival curves were created by fitting surviving fractions to the linear quadraticequation. D, Flow cytometric analysis showing cell apoptosis in the A20 OE CNE2-IR cells, A20 KD CNE2 cells, and their respective control cells 72 hoursafter 6 Gy ionizing radiation. Three experiments were done; mean, SDs, and statistical significance are denoted; �� , P < 0.001. Vector, transfected with anempty vector.

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OE abrogated the effect of miR-125b inhibitor on NF-kB activityin the radiosensitive CNE2 cells, and A20 KD restored activityof NF-kB decreased by miR-125b mimic in the radioresistantCNE2-IR cells (Fig. 6D). Collectively, these results demonstratethat miR-125b regulates activity of the NF-kB signaling pathwayby targeting A20.

Next, we determined whether NF-kBmediates miR-125b/A20-regulating NPC cell response. We observed that either IkBaOE orNF-kB inhibitor BAY11-7082 significantly abolished radioresis-tance induced by A20 KD in the radioresistant CNE2 cells,whereas NF-kB p65 OE restored radioresistance reduced byA20 OE in the radiosensitive CNE2-IR cells (Fig. 7A–C). Theseresults demonstrate that NF-kB signaling mediates miR-125b/A20-regulating NPC cell radioresponse.

Levels of miR-125b, A20, and p-p65 are correlated in humanNPC biopsies

Because our data demonstrate that miR-125b regulates NPCcell radioresponse through targeting A20/NF-kB, we next deter-mined whether the levels of miR-125b, A20, and p-p65 werecorrelated in NPC biopsies. Our IHC analysis showed that A20expression was significantly lower while p-p65 was significantlyhigher in the radioresistant NPCs than that in the radiosensitiveNPCs (Fig. 1C; Supplementary Table S3). Correlation analysesrevealed that miR-125b level was negatively associated with A20

level (r ¼ �0.61, P < 0.001), whereas positively associated withp-p65 level (r ¼ 0.45, P < 0.001), and A20 level was negativelyassociated with p-p65 level (r ¼ �0.38, P < 0.001). The resultsindicate that NF-kB signaling might mediate miR-125b/A20-regulating the radioresponse of clinical NPCs.

DiscussionRadioresistance is the main obstacle in the clinical manage-

ment of NPC (2, 3). Investigating the role of miRNAs in radio-resistance is a promising avenue given their ability to regulatemultiple oncogenic processes including response to therapy (41).In this study, we focused on miR-125b, one of upregulatedmiRNAs in the radioresistant NPC cells, because the functionand mechanism of miR-125b in NPC radioresistance are unclear.We found that miR-125b increased NPC cell radioresistance bothin vitro and in vivo. The finding is clinically relevant, given ourdiscovery that miR-125b was frequently upregulated in radio-resistant NPCs, and its increment was correlated with NPC radio-resistance and poor patient survival, outlining a potential markerfor predicting the radioresponse and prognosis of NPC patients,and suggesting its considerable potential in clinical NPCradiosensitization.

As miRNAs exert their roles through inhibiting target mRNAtranslation, thus identification of miR-125b target genes is a key

100

nmol

/L50

nm

ol/L

Con

trol

0 Gy 2 GyCNE2-IR/A20-OE+miR125b Mimic

A B

4 Gy 6 Gy 8 Gy 10 Gy

CNE2-IR/A20-OEMimic controlmiRNA-125b mimic (50 nmol/L)miRNA-125b mimic (100 nmol/L)

CNE2/A20-KDInhibitor control

miRNA-125b inhibitor (100 nmol/L)miRNA-125b inhibitor (50 nmol/L)

Surv

ival

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tion

0.1

1

0.01

0.0010 2 4

Dose (Gy)

P < 0.05



6 8 10

Surv

ival

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tion

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0.0010 2 4

Dose (Gy)

P < 0.05



6 8 10

Control

Control

50 nmol/L

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mol/L

100 n

mol/L

35

30

Apo

ptot

ic ra

te (%

)

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20

15

10

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0

miR-125b Mimic miR-125b InhibitorCNE2-IR/A20-OE CNE2/A20-KD

100

nmol

/L50

nm

ol/L

Con

trol

0 Gy 2 GyCNE2/A20-KD+miR125b inhibitor

4 Gy 6 Gy 8 Gy 10 Gy

CNE2/A20-KD

CNE2-IR/A20-OEmiR-125b mimic

Inhibitor controlmiR-125b Inhibitor

50 nmol/L 100 nmol/L

Mimic control 50 nmol/L 100 nmol/L

Figure 4.

MiR-125b increases NPC cell radioresistance through targeting A20. A, A clonogenic survival assay showing the radioresponse of A20 OE CNE2-IR cellstransfected with miR-125b inhibitor, A20 KD CNE2 cells transfected with miR-125b mimic, and their control cells. Left, cells were irradiated with a rangeof 0 to 10 Gy radiation doses, and colonies that formed after incubation of 12 days were stained with crystal violet and photographed; right, dose survivalcurves were created by fitting surviving fractions to the linear quadratic equation. B, Flow cytometric analysis showing cell apoptosis in the A20 KD CNE2 cellstransfected with miR-125b inhibitor, A20 OE CNE2-IR cells transfected with miR-125b mimic, and their control cells 72 hours after 6 Gy ionizing radiation.Three experiments were done; mean, SDs, and statistical significance are denoted; �� , P < 0.001. Vector, transfected with an empty vector.



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step for understanding the mechanism of miR-125b–regulatingNPC radioresistance. In this study, we confirm that A20 is a directtarget ofmiR-125b inNPC cells, andmiR-125b regulates NPC cellradioresponse through targeting A20. Numerous studies haverevealed that A20, an ubiquitin-editing enzyme, negatively reg-ulates activity of theNF-kB signaling pathway (22–26). Activationof the NF-kB signaling pathway not only plays a crucial role in the

development and progression of NPC (14–16), but also conferstumor resistance to radiotherapy (17–20). Therefore, we investi-gated whether NF-kB mediates miR-125b/A20-regulating NPCcell radioresponse. We observed that miR-125bmimic or A20 KDenhanced, whereas miR-125b inhibitor or A20 OE reduced activ-ity of NF-kB in NPC cells; A20 OE abrogated activity of NF-kBinduced by miR-125b inhibitor, and A20 KD restored activity of

CNE2-IR CNE2-IR/vector

CNE2-IR/A20-OE2CNE2-IR/A20-OE1

CNE2 CNE2/scr-shRNA

CNE2/A20-KD2CNE2/A20-KD1

CNE2-IR

CNE2-IR/vector

0.6

CNE2-IRCNE2-IR/vectorCNE2-IR/A20-OE1CNE2-IR/A20-OE2

CNE2-IRBlank Vector A20-OE1 A20-OE2

CNE2Blank src-shRNA A20-KD1 A20-KD2

A20

p-p65

TUNEL

gH2AX

0.5

0.4

0.3

0.2

0.1

0.0Xeno

graf

t tum

or w

eigh

t (g) 3.5

00

500

1,0001,5002,000

2,5003,000

3,500

Tum

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m3 )

3 6 9 12 15 18 21Time after irradiation (day)

3.0

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CNE2CNE2/scr-shRNACNE2/A20-KD1CNE2/A20-KD2

00

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3 6 9 12 15 18 21Time after irradiation (day)

CNE2-IRCNE2-IR/vectorCNE2-IR/A20-OE1CNE2-IR/A20-OE2

CNE2CNE2/scr-shRNACNE2/A20-KD1CNE2/A20-KD2

Xeno

graf

t tum

or w

eigh

t (g)

CNE2-IR/A20-OE1

CNE2-IR/A20-OE2

CNE2

CNE2/scr-shRNA

CNE2/A20-KD1

CNE2/A20-KD2

8

6

4

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8 40

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0A20 p-p65 TUNEL gH2AX

A20

Exp

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p-p6

5 Ex

pres

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ce

Perc

enta

ge o

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Perc

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fgH

2AX-

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cells

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0A20 p-p65 TUNEL gH2AX

A20

Exp

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p-p6

5 Ex

pres

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sor

ce

Perc

enta

ge o

fap

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ells

(%)

Perc

enta

ge o

fpo

sitiv

e gH

2AX

cells

BlankVectorA20-OE1A20-OE2

Blanksrc-shRNAA20-KD1A20-KD2

A B

C

Figure 5.

A20 decreases NPC cell radioresistance in vivo. A, The gross appearance (left) and growth and weight (right) of subcutaneous xenograft tumors generatedbyA20OECNE2 cells and control cells 21 days after 6Gy irradiation (n¼ 5 each group).B,Thegross appearance (left) andgrowth andweight (right) of subcutaneousxenograft tumors generated by A20 KD CNE2 cells and control cells 21 days after 6 Gy irradiation (n ¼ 5 each group). C, Immunohistochemistry showingthe expression of A20, p-p65, and gH2AX and TUNEL-detected apoptotic cells in the subcutaneous xenograft tumors generated by A20 OE CNE2-IR cells,A20 KD CNE2 cells, and their respective control cells 21 days after 6 Gy irradiation. Original magnification,�400. Mean, SDs, and statistical significance are denoted;�� , P < 0.01; �� , P < 0.01. Vector, transfected with an empty vector.

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CNE2A B

C

D

CNE2

1.0 1.0 1.0

1.01.0

1.0 1.0

1.01.0

1.0

1.0

1.0

1.0

1.01.0 0.94 0.97

1.78 1.68

0.690.7

1.67 1.73

0.96 1.07

1.37 1.71

0.59 0.77 1.68 1.63

0.80.42

0.85 0.79

0.790.69

2.912.24

0.5

0.9

0.71

0.82

1.25 0.63 1.0 1.56 5.01A20

p-IKK-a/b

p-IkBa

IkBa

p-p65

p65

b-Actin

CNE2-IR

BlankInhibitorcontrol

miR-125binhibitor vector A20-OE

IKK-a/b2.540.921.0 1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.07

1.07

1.91

0.84

2.57

0.58 1.34

0.77 0.30

2.86

0.721.071.0

1.0 1.11.07

1.0

1.0

1.61 2.79

1.171.17

1.02 0.57

0.920.92

CNE2-IR

CNE2-IRCNE2-IR

ns

ns

ns

ns

25

Rel

ativ

e lu

cife

rase

act

ivity

(fold

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Rel

ativ

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)

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Rel

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Blank

DAPI

p-p65

Merge

DAPI

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Merge

Controlmimic

miR-125bMimic

CNE2-IR/A20-OEmiR-125b

MimicMimiccontrolBlank

DAPI

p-p65

Merge

CNE2/A20-KDmiR-125bInhibitor

InhibitorcontrolBlank

DAPI

p-p65

Merge

p-p65

CNE2-IR/A20-OE

Blank

Mimic

contro

l

miR-12

5b M

imic

p65

A20

Tubulin1.0

1.0

1.0 1.07 2.14

0.810.82

1.19 0.68

p-p65

CNE2/A20-KD

Blank

Inhibitor c

ontrol

miR-12

5b In

hibitor

p65

A20

Tubulin1.0

1.0

1.0 1.03 0.24

0.961.16

0.91 2.51

scr-shRNA A20-KD

0

pGL6-TA+pRL-TKpNF-kB-TA-Luc+pRL-TK

CNE2-IR/OE

ns

ns

25

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15

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Mimic

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-125b

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Inhibitor c

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5b In

hibitor

pGL6-TA+pRL-TKpNF-kB-TA-Luc+pRL-TK

CNE2/A20-KDpGL6-TA+pRL-TKpNF-kB-TA-Luc+pRL-TK

CNE2pGL6-TA+pRL-TKpNF-kB-TA-Luc+pRL-TK

Blank

Blank

Mimic

contro

l

Inhibitor c

ontrol

miR-125b

mim

ic

src-sh

RNA

A20-K

D1

A20-K

D2

A20-O

E2

A20-O

EBlan

kVec

tor

miR-125b

Inhibito

r

Inhibitor c

ontrol

A20-K

DBlan

k

scr-s

hRNA

miR-125b

Mim

ic

Mimic

contro

l

A20-O

E1

Vector

miR-125b

inhibito

r

Figure 6.

MiR-125b regulates the activity of theNF-kB signaling pathway by targetingA20 inNPC cells.A,Western blot analysis showing the levels of p-IKKa/b, p-IkBa, p-p65,IKKa/b, IkBa, and p65 in the miR-125b mimic–transfected or A20 KD CNE2 cells, miR-125b inhibitor–transfected or A20 OE CNE2-IR cells, and theirrespective control cells. B, A luciferase reporter assay showing p65 (RelA) transcriptional activity in the A20 OE or miR-125 inhibitor–transfected CNE2-IR cells,A20 KD or miR-125b mimic–transfected CNE2 cells, and their respective control cells. C, Representative immunofluorescent staining showing the nucleartranslocation of p-p65 in themiR-125b inhibitor–transfected orA20OECNE2-IR cells, miR-125bmimic–transfected or A20KDCNE2 cells, and their respective controlcells. D, Western blot analysis showing p-p65 levels (left), representative immunofluorescent staining showing the nuclear translocation of p-p65 (middle),and luciferase reporter assay showingp65 transcriptional activity (right) in theA20OECNE2-IR cells transfectedwithmiR-125bmimic, A20KDCNE2 cells transfectedwith miR-125b inhibitor, and their respective control cells. Three experiments were done; mean, SDs, and statistical significance are denoted; �� , P < 0.001; ns,no significance. Vector, transfected with an empty vector.



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NF-kB decreased by miR-125b mimic in the NPC cells, demon-strating that miR-125b activates NF-kB by targeting A20. Wefurther showed that IkBa OE or BAY11-7082 could abolishradioresistance induced by A20 KD, whereas p65 (RelA) OErestored radioresistance reduced by A20 OE in the NPC cells. Inthe clinical NPC samples, p-p65 level was increased in the radio-resistant NPCs relative to radiosensitive NPCs, and negatively

associated with A20 level while positively associated withmiR-125b level. Taken together, our results demonstrate that NF-kB mediates miR-125b/A20-regulating NPC cell radioresponse.

Numerous studies have indicated that A20 is involved in thepathogenesis of various types of human tumors (30–35). How-ever, the roles of A20 in tumor radioresistance are unclear. In thisstudy, we observed that A20 was decreased in the radioresistant

2 mg

1 mg

0 mg

0 Gy 2 GyCNE2/A20-KD+IkBa

A4 Gy 6 Gy 8 Gy

2 mg

1 mg

0 mg

0 Gy 2 Gy

CNE2-IR/A20-OE+p65C4 Gy 6 Gy 8 Gy

0 mm

ol/L

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/L

0 Gy 2 GyCNE2/A20-KD+BAY 11-7082B

4 Gy 6 Gy 8 GySu

rviv

al fr

actio

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0.1

1

CNE2

A20-KDA20-KD+1 mg IkBaA20-KD+2 mg IkBa

CNE2A20-KDA20-KD+5 mmol/L BAY 11-7082A20-KD+10 mmol/L BAY 11-7082

CNE2-IRCNE2-IR/A20-OE+p65

1 mg 2 mg0 mg

CNE2/A20-KD+IkBa1 mg 2 mg0 mg

CNE2/A20-KD+BAY 11-70820 mmol/L 5 mmol/L 10 mmol/L

A20-OEA20-OE+1 mg p65A20-OE+2 mg p65

0.01

0.0010 2 4

Dose (Gy)

P < 0.05

RPF(A20-KD+1 mg IkBa) = 0.62RPF(A20-KD+2 mg IkBa) = 0.50

6 8

Surv

ival

frac

tion

0.1

1

0.01

0.0010 2 4

Dose (Gy)

P < 0.05

RPF(A20-KD+5 mmol/L BAY 11-7082) = 0.895RPF(A20-KD+10 mmol/L BAY 11-7082) = 0.590

6 8

Surv

ival

frac

tion

0.1

1

0.01

0.0010 2 4

Dose (Gy)

P < 0.05

RPF(A20-OE+2 mg p65) = 1.18RPF(A20-OE+4 mg p65) = 1.30

6 80

5

10

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35

Apo

ptot

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)

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Apo

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)

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1015

20

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35

Apo

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te (%

)

Figure 7.

NF-kB mediates miR-125b/A20-regulating NPC cell radioresponse. A, A clonogenic survival assay showing the radioresponse of A20 KD CNE2 cellstransfected with plasmid expressing IkBa and control cells (left and middle), and flow cytometric analysis showing cell apoptosis of A20 KD CNE2 cellstransfected with plasmid expressing IkBa and control cells (right). B, A clonogenic survival assay showing the radioresponse of A20 KD CNE2 cells treatedwith BAY 11-7082 and control cells(left and middle), and flow cytometric analysis showing cell apoptosis of A20 KD CNE2 cells treated with BAY 11-7082and control cells (right). C, A clonogenic survival assay showing the radioresponse of A20 KD CNE2-IR cells transfected with plasmid expressing p65 andcontrol cells (left and middle), and flow cytometric analysis showing cell apoptosis of A20 OE CNE2-IR cells transfected with plasmid expressing p65 and controlcells (right). Three experiments were done; mean, SDs, and statistical significance are denoted; � , P < 0.05; �� , P < 0.01.

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NPCs relative to radiosensitive NPCs; A20 OE reduced while A20KD enhanced NPC cell radioresistance in vitro and in vivo. Theresults strongly demonstrate that low A20 expression increasesNPC radioresistance, suggesting its considerable potential inclinical NPC radiosensitization. To our knowledge, this is firstreported that A20 regulates tumor radioresponse.

Although the A20/NF-kB signaling axis seems to largelyaccount for the radioresistant phenotype of NPC cells inducedbymiR-125, indeed a singlemiRNA can targetmultiplemRNAs toregulate gene expression (41). Therefore, there might be othermolecules such as BAK1, PPP1CA, and p53 (42–46), which arealso targeted by miR-125b in NPC cells. We also observed thatmiR-125b negatively modulated p53 expression in NPC cells, butit still inhibited cell apoptosis in p53 KDNPC CNE2 cell line thatwas established previously by us (47). The results suggest thatmiR-125b increases NPC cell radioresistance by p53-independentmanner, 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-125bexpression by using miR-125b antagomir enhanced NPC radio-sensitivity in NPC xenografts. Nucleic-acid drugs, such as miR-NAs, can be directly synthesized and modified to be more lipo-philic that improves penetration. Such modification includescholesterylation. Our delivery of miR-125b antagomir, choles-terylated miRNA inhibitor, successfully increased NPC radiosen-sitivity in intratumoral injectionmodel, suggesting thatmiR-125bantagomir has a potential for further drug development.

In summary, our data demonstrate that miR-125b is frequentlyupregulated in the radioresistant NPCs and is an independentpredictor for NPC survival; miR-125b regulates NPC cell radio-sensitivity in vitro and in vivo through targeting A20 NF-kB sig-naling pathway; A20 is frequently downregulated in the radio-resistant 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 criticalregulators of NPC radioresponse and suggests that targeting themiR-125b/A20/NF-kB signaling axis is a promising approach forenhancing NPC sensitivity to radiotherapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L.-N. Li, T. Xiao, Z.-Q. XiaoDevelopment of methodology: L.-N. Li, T. XiaoAcquisition 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. LuAnalysis 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. LiWriting, review, and/or revision of the manuscript: T. Xiao, Z.-Q. XiaoStudy supervision: Z.-Q. XiaoOther (processing tables and figures): W. Huang

AcknowledgmentsWe thank Dr. Tiebang Kang (The Cancer Center, Sun Yat-sen University,

China) for providing pcDNA3.1-p65/RelA and pcDNA3.1-IkBa expression andcontrol plasmids.

Grant SupportThis work was supported by Major Program of the National Natural Science

Foundation of China (81230053, to Z.-Q. Xiao), National Basic ResearchProgram of China (2013CB910502, to Z.-Q. Xiao), and the National NaturalScience Foundation of China (81472801, to X.-H. Li; 81672687, to Z.-Q. Xiao).

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 1, 2017; revised June 1, 2017; accepted June 19, 2017;published OnlineFirst July 11, 2017.

References1. LoKW, ToKF,HuangDP. Focus onnasopharyngeal carcinoma. CancerCell

2004;5:423–8.2. KristensenCA, Kjaer-Kristoffersen F, SapruW, Berthelsen AK, Loft A, Specht

L. Nasopharyngeal carcinoma. Treatment planning with IMRT and 3Dconformal radiotherapy. Acta Oncol 2007;46:214–20.

3. Lee AW, PoonYF, FooW, Law SC, Cheung FK, ChanDK, et al. Retrospectiveanalysis of 5037 patients with nasopharyngeal carcinoma treated during1976–1985: overall survival and patterns of failure. Int J Radiat Oncol BiolPhys 1992;23:261–70.

4. Garzon R, Calin GA, Croce CM. MicroRNAs in cancer. Annu Rev Med2009;60:167–79.

5. Huang X, Taeb S, Jahangiri S, Emmenegger U, Tran E, Bruce J, et al. miRNA-95 mediates radioresistance in tumors by targeting the sphingolipidphosphatase SGPP1. Cancer Res 2013;73:6972–86.

6. Wang P, Zhang J, Zhang L, Zhu Z, Fan J, Chen L, et al. MicroRNA 23bregulates autophagy associated with radioresistance of pancreatic cancercells. Gastroenterology 2013;145:1133–43.

7. Ke G, Liang L, Yang JM, Huang X, Han D, Huang S, et al. MiR-181a confersresistance of cervical cancer to radiation therapy through targeting the pro-apoptotic PRKCD gene. Oncogene 2013;32:3019–27.

8. Gwak HS, Kim TH, Jo GH, Kim YJ, Kwak HJ, Kim JH, et al. Silencing ofmicroRNA-21 confers radio-sensitivity through inhibition of the PI3K/AKTpathway and enhancing autophagy in malignant glioma cell lines. PLoSOne 2012;7:e47449.

9. Oh JS, Kim JJ, Byun JY, Kim IA. Lin28-let7 modulates radiosensitivity ofhuman cancer cells with activation of K-Ras. Int J Radiat Oncol Biol Phys2010;76:5–8.

10. QuC, Liang Z,Huang J, ZhaoR, SuC,WangS, et al.MiR-205determines theradioresistance of human nasopharyngeal carcinoma by directly targetingPTEN. Cell Cycle 2012;11:785–96.

11. Grosso S, Doyen J, Parks SK, Bertero T, Paye A, Cardinaud B, et al. MiR-210promotes a hypoxic phenotype and increases radioresistance in humanlung cancer cell lines. Cell Death Dis 2013;4:e544.

12. Li G, Liu Y, Su Z, Ren S, Zhu G, Tian Y, et al. MicroRNA-324-3p regulatesnasopharyngeal carcinoma radioresistance by directly targeting WNT2B.Eur J Cancer 2013;49:2596–607.

13. Aggarwal BB, Sung B. NF-kB in cancer: a matter of life and death. CancerDiscov 2011;1:469–71.

14. Verhoeven RJ, Tong S, Zhang G, Zong J, Chen Y, Jin DY, et al. NF-kBsignaling regulates expression of Epstein-Barr virus BART microRNAs andlong noncoding RNAs in nasopharyngeal carcinoma. J Virol 2016;90:6475–88.

15. ZhuDD, Zhang J, DengW, Yip YL, LungHL, Tsang CM, et al. Significance ofNF-kB activation in immortalization of nasopharyngeal epithelial cells. IntJ Cancer 2016;138:1175–85.

16. Chung GT, Lou WP, Chow C, To KF, Choy KW, Leung AW, et al. Consti-tutive activation of distinct NF-kB signals in EBV-associated nasopharyn-geal carcinoma. J Pathol 2013;231:311–22.

17. Ahmed KM, Zhang H, Park CC. NF-kB regulates radioresistance mediatedby b1-integrin in three-dimensional culture of breast cancer cells. CancerRes 2013;73:3737–48.

18. Veuger SJ, Hunter JE, Durkacz BW. Ionizing radiation-induced NF-kappaBactivation requires PARP-1 function to confer radioresistance. Oncogene2009;28:832–42.



Retrac

ted N

ovem

ber 2

, 201

8





19. Ahmed KM, Li JJ. NF-kappa B-mediated adaptive resistance to ionizingradiation. Free Radic Biol Med 2008;44:1–13.

20. Deorukhkar A, Krishnan S. Targeting inflammatory pathways for tumorradiosensitization. Biochem Pharmacol 2010;80:1904–14.

21. Liu S, Chen ZJ. Expanding role of ubiquitination in NF-kB signaling. CellRes 2011;21:6–21.

22. Wertz IE, O'Rourke KM, Zhou H, Eby M, Aravind L, Seshagiri S, et al.Deubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature 2004;430:694–9.

23. Shembade N,Ma A, Harhaj EW. Inhibition of NF-kappaB signaling by A20through disruption of ubiquitin enzyme complexes. Science 2010;327:1135–9.

24. Hymowitz SG, Wertz IE. A20: from ubiquitin editing to tumour suppres-sion. Nat Rev Cancer 2010;10:332–41.

25. Vereecke L, Beyaert R, van Loo G. The ubiquitin-editing enzyme A20(TNFAIP3) is a central regulator of immunopathology. Trends Immunol2009;30:383–91.

26. Shembade N, Harhaj EW. Regulation of NF-kB signaling by the A20deubiquitinase. Cell Mol Immunol 2012;9:123–30.

27. Kim SW, Ramasamy K, BouamarH, Lin AP, JiangD, Aguiar RC.MicroRNAsmiR-125a and miR-125b constitutively activate the NF-kB pathway bytargeting the tumor necrosisfactor alpha-induced protein 3 (TNFAIP3,A20). Proc Natl Acad Sci U S A 2012;109:7865–70.

28. Parisi C, Napoli G, Amadio S, Spalloni A, Apolloni S, Longone P, et al.MicroRNA-125b regulatesmicroglia activation andmotor neuron death inALS. Cell Death Differ 2016;23:531–41.

29. Liu Z, Smith KR, Khong HT, Huang J, Ahn EE, Zhou M, et al. miR-125bregulates differentiation and metabolic reprogramming of T cell acutelymphoblastic leukemia by directly targeting TNFAIP3. Oncotarget2016;7:78667–79.

30. Troppan K, Hofer S, Wenzl K, Lassnig M, Pursche B, Steinbauer E, et al.Frequent down regulation of the tumor suppressor gene TNFAIP3 inmultiple myeloma. PLoS One 2015;10:e0123922.

31. Langsch S, Baumgartner U, Haemmig S, Schlup C, Sch€afer SC, BerezowskaS, et al.miR-29bmediatesNF-kB signaling inKRAS-inducednon-small celllung cancers. Cancer Res 2016;76:4160–9.

32. Kato M, Sanada M, Kato I, Sato Y, Takita J, Takeuchi K, et al. Frequentinactivation of TNFAIP3 in B-cell lymphomas. Nature 2009;459:712–6.

33. Hjelmeland AB, Wu Q, Wickman S, Eyler C, Heddleston J, Shi Q, et al.Targeting TNFAIP3 decreases glioma stem cell survival and tumor growth.PLoS Biol 2010;8:e1000319.

34. Wang CM, Wang Y, Fan CG, Xu FF, Sun WS, Liu YG, et al. miR-29c targetsTNFAIP3, inhibits cell proliferation and induces apoptosis in hepatitis Bvirus-related hepatocellular carcinoma. Biochem Biophys Res Commun2011;411:586–92.

35. Bellai AC, Olson JJ, Yang X, Chen ZJ, Hao C. TNFAIP3 ubiquitin ligase-mediated polyubiquitination of RIP1 inhibits caspase-8 cleavage andTRAIL- induced apoptosis in glioblastoma. Cancer Discov 2012;2:140–55.

36. Shanmugaratnam K, Sobin LH. The World Health Organization histolog-ical classification of tumours of the upper respiratory tract and ear.A commentary on the second edition. Cancer 1993;71:2689–97.

37. Pan J, Xu Y,Qiu S, Zong J, GuoQ,Zhang Y, et al. AComparison between theChinese 2008 and the 7th editionAJCC staging systems for nasopharyngealcarcinoma. Am J Clin Oncol 2015;38:189–96.

38. Qu JQ, Yi HM, Ye X, Zhu JF, Yi H, Li LN, et al. MiRNA-203 reducesnasopharyngeal carcinoma radioresistance by targeting IL8/AKT signaling.Mol Cancer Ther 2015;14:2653–64.

39. Feng XP, Yi H, Li MY, Li XH, Yi B, Zhang PF, et al. Identification ofbiomarkers for predicting nasopharyngeal carcinoma response to radio-therapy by proteomics. Cancer Res 2010;70:3450–62.

40. Gudkov AV, Komarova EA. The role of p53 in determining sensitivity toradiotherapy. Nat Rev Cancer 2003;3:117–29.

41. SelbachM, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N.Widespread changes in protein synthesis induced by microRNAs. Nature2008;455:58–63.

42. Busch S, Auth E, Scholl F, Huenecke S, Koehl U, Suess B, et al. 5-lipox-ygenase is a direct target of miR-19a-3p and miR-125b-5p. J Immunol2015;194:1646–53.

43. Wu JG, Wang JJ, Jiang X, Lan JP, He XJ, Wang HJ, et al. MiR-125b promotescell migration and invasion by targeting PPP1CA-Rb signal pathways ingastric cancer, resulting in a poor prognosis. Gastric Cancer 2015;18:729–39.

44. Wang YD, Cai N, Wu XL, Cao HZ, Xie LL, Zheng PS. OCT4 promotestumorigenesis and inhibits apoptosis of cervical cancer cells by miR-125b/BAK1 pathway. Cell Death Dis 2013;4:e760.

45. Amir S, Ma AH, Shi XB, Xue L, Kung HJ, Devere White RW. OncomirmiR-125b suppresses p14 (ARF) to modulate p53-dependent andp53-independent apoptosis in prostate cancer. PLoS One 2013;8:e61064.

46. Ahuja D, Goyal A, Ray PS. Interplay between RNA-binding protein HuRand microRNA-125b regulates p53 mRNA translation in response togenotoxic stress. RNA Biol 2016;13:1152–65.

47. Sun Y, Yi H, Yang Y, Yu Y, Ouyang Y, Yang F, et al. Functional character-ization of p53 in nasopharyngeal carcinoma by stable shRNA expression.Int J Oncol 2009;34:101710–27.

48. Lv P, Wang Y, Ma J, Wang Z, Li JL, Hong CS, et al. Inhibition of proteinphosphatase 2A with a small molecule LB100 radiosensitizes nasopha-ryngeal carcinoma xenografts by inducing mitotic catastrophe and block-ing DNA damage repair. Oncotarget 2014;5:7512–24.

49. He JH, Liao XL, WangW, Li DD, ChenWD, Deng R, et al. Apogossypolone,a small-molecule inhibitor of Bcl-2, induces radiosensitization of naso-pharyngeal carcinoma cells by stimulating autophagy. Int J Oncol2014;45:1099–108.

50. Zhang L, Yang L, Li JJ, Sun L. Potential use of nucleic acid-based agents inthe sensitizationof nasopharyngeal carcinoma to radiotherapy.Cancer Lett2012;323:1–10.


Li et al.

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ovem

ber 2

, 201

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Retraction

Retraction: MiR-125b IncreasesNasopharyngeal Carcinoma RadioresistanceBy Targeting A20/NF-kB Signaling PathwayLi-Na Li, Ta Xiao, Hong-Mei Yi, Zhen Zheng, Jia-Quan Qu,Wei Huang, Xu Ye, Hong Yi, Shan-Shan Lu, Xin-Hui Li, andZhi-Qiang Xiao

This article (1) has been retracted at the request of the authors. The Journal wasmade aware that the article contains extensive duplication of text and datapreviously published in a Cell Death & Disease article (2). In addition to textduplication, data reuse was found in Figures 1, 3, and 6, and SupplementaryTable S4. The authors deeply regret any inconveniences or challenges resultingfrom the publication and subsequent retraction of this article.

A copy of this retraction notice was sent to the last known e-mail addresses for the11 authors. All authors agreed to the retraction.

References1. Li LN, Xiao T, Yi HM, Zheng Z, Qu JQ, Huang W, et al. MiR-125b increases nasopharyngeal

carcinoma radioresistance by targeting A20/NF-kB signaling pathway. Mol Cancer Ther 2017;16:2094–106.

2. Zheng Z, Qu JQ, Yi HM, Ye X, Huang W, Xiao T, et al. MiR-125b regulates proliferation andapoptosis of nasopharyngeal carcinoma by targeting A20/NF-kB signaling pathway. Cell DeathDis 2017;8:e2855.

Published online November 2, 2018.doi: 10.1158/1535-7163.MCT-18-0938�2018 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 24(11) November 20182490

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-18-0938&domain=pdf&date_stamp=2018-10-22

2017;16:2094-2106. Published OnlineFirst July 11, 2017.Mol Cancer Ther Li-Na Li, Ta Xiao, Hong-Mei Yi, et al.

B Signaling Pathwayκby Targeting A20/NF-MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance

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MiR-125b Increases Nasopharyngeal Carcinoma ......Small Molecule Therapeutics MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance by Targeting A20/NF-kB Signaling Pathway Li-Na