Top Banner
MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Ye a,1 , Ning-xia Sun a,1 , Yan Ma a,b , Qian Zhao a , Qing Zhang a , Chen Xu a , Shao-bing Wang b , Shu-han Sun b , Fang Wang b,, Wen Li a,a Department of Obstetrics & Gynaecology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China b Department of Medical Genetics, Second Military Medical University, Shanghai, China article info Article history: Received 8 December 2014 Revised 21 January 2015 Accepted 28 January 2015 Available online 7 February 2015 Edited by Tamas Dalmay Keywords: Cervical cancer Radiosensitivity Microarray MicroRNA-145 Helicase-like transcription factor abstract In our study, transcriptome microarrays are used to identify differentially expressed miRNAs and mRNAs in cervical cancer specimens. We find that microRNA-145 (miR-145) expression is signifi- cantly decreased in cervical cancer tissues and cell lines, and is associated with advanced cancer stages, large tumor size and moderate/poor differentiation. We show that miR-145 targets the DNA damage repair-associated gene Helicase-like transcription factor (HLTF), which is involved in radio-resistance. Moreover, miR-145 over-expression in cervical cancer cells enhances radiosensitiv- ity in vitro and in vivo. These results indicate that targeting miR-145 may be a novel radiosensitizing strategy for cervical cancer. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction MicroRNAs (miRNAs) are a class of small non-coding RNAs that can regulate gene expression through the degradation or transla- tional inhibition of target mRNAs [1]. An increasing number of miRNAs has been demonstrated to been involved in the initiation and progression of various human malignancies [2] and potentially represent novel diagnostic and prognostic markers [3,4]. Moreover, recent evidence has linked several miRNAs with treatment response, such as let-7e [5], miR-375 [6] and miR-200c [7], which play important roles in radiation or chemotherapy sensitivity. Cervical cancer is one of the leading gynecological malignancies worldwide [8]. Previous studies have reported that many miRNAs can act as oncogenes (e.g., miR-182, miR-205) [9,10] or tumor suppressors (e.g., miR-7, miR-214) [11,12] in cervical cancer. How- ever, our understanding of the potential roles of miRNAs in the prognosis evaluation and treatment response of cervical cancer is still limited. Combining miRNA expression information with clini- copathological characteristics and identifying the lead target mRNAs are helpful in guiding the study of the functions and mech- anisms of abnormally expressed miRNAs in cervical cancer. In our study, to screen aberrant miRNAs and predict the corre- sponding target mRNAs, high-throughput transcriptome micro- arrays were used to detect differentially expressed miRNAs and mRNAs synchronously in cervical cancer tissues compared with adjacent non-tumor tissues. From our microarray results, we investigated the most significantly down-regulated (>4-fold) miR- NA (Hsa-miR-145-5p, miR-145) in clinical specimens and ana- lyzed the association between miR-145 expression levels and specific clinicopathological characteristics. Then, we combined microarray analyses and bioinformatic predictions to screen can- didate target mRNAs of miR-145 effectively and accurately. Heli- case-like transcription factor (HLTF) was the best candidate target gene, and it was found to confer radiation resistance in cervical cancer in a recent study [13]. Moreover, we further demonstrated that miR-145 over-expression in cervical cancer cells could enhance radiosensitivity in vitro and in vivo, which showed a additional anticancer function of miR-145 in cervical cancer. These findings might help establish new strategies for therapy decision-making and improving therapeutic effect of cervical cancer. http://dx.doi.org/10.1016/j.febslet.2015.01.037 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Corresponding authors at: Department of Medical Genetics, Second Military Medical University, Shanghai, 800 Xiang Yin road, Shanghai 200433, China. Fax: +86 021 81871053 (F. Wang). Department of Obstetrics & Gynaecology, Shanghai Changzheng Hospital, Second Military Medical University, No. 415 Fengyang Rd, Shanghai 200003, China. Fax: +86 21 81886711 (W. Li). E-mail addresses: [email protected] (F. Wang), [email protected] (W. Li). 1 These authors contributed equally to this work. FEBS Letters 589 (2015) 702–709 journal homepage: www.FEBSLetters.org
8

MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

May 29, 2019

Download

Documents

trinhtruc
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Page 1: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

FEBS Letters 589 (2015) 702–709

journal homepage: www.FEBSLetters .org

MicroRNA-145 contributes to enhancing radiosensitivity of cervicalcancer cells

http://dx.doi.org/10.1016/j.febslet.2015.01.0370014-5793/� 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

⇑ Corresponding authors at: Department of Medical Genetics, Second MilitaryMedical University, Shanghai, 800 Xiang Yin road, Shanghai 200433, China. Fax: +86021 81871053 (F. Wang). Department of Obstetrics & Gynaecology, ShanghaiChangzheng Hospital, Second Military Medical University, No. 415 Fengyang Rd,Shanghai 200003, China. Fax: +86 21 81886711 (W. Li).

E-mail addresses: [email protected] (F. Wang), [email protected] (W. Li).1 These authors contributed equally to this work.

Chen Ye a,1, Ning-xia Sun a,1, Yan Ma a,b, Qian Zhao a, Qing Zhang a, Chen Xu a, Shao-bing Wang b,Shu-han Sun b, Fang Wang b,⇑, Wen Li a,⇑a Department of Obstetrics & Gynaecology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, Chinab Department of Medical Genetics, Second Military Medical University, Shanghai, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 December 2014Revised 21 January 2015Accepted 28 January 2015Available online 7 February 2015

Edited by Tamas Dalmay

Keywords:Cervical cancerRadiosensitivityMicroarrayMicroRNA-145Helicase-like transcription factor

In our study, transcriptome microarrays are used to identify differentially expressed miRNAs andmRNAs in cervical cancer specimens. We find that microRNA-145 (miR-145) expression is signifi-cantly decreased in cervical cancer tissues and cell lines, and is associated with advanced cancerstages, large tumor size and moderate/poor differentiation. We show that miR-145 targets theDNA damage repair-associated gene Helicase-like transcription factor (HLTF), which is involved inradio-resistance. Moreover, miR-145 over-expression in cervical cancer cells enhances radiosensitiv-ity in vitro and in vivo. These results indicate that targeting miR-145 may be a novel radiosensitizingstrategy for cervical cancer.� 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

MicroRNAs (miRNAs) are a class of small non-coding RNAs thatcan regulate gene expression through the degradation or transla-tional inhibition of target mRNAs [1]. An increasing number ofmiRNAs has been demonstrated to been involved in the initiationand progression of various human malignancies [2] and potentiallyrepresent novel diagnostic and prognostic markers [3,4]. Moreover,recent evidence has linked several miRNAs with treatmentresponse, such as let-7e [5], miR-375 [6] and miR-200c [7], whichplay important roles in radiation or chemotherapy sensitivity.

Cervical cancer is one of the leading gynecological malignanciesworldwide [8]. Previous studies have reported that many miRNAscan act as oncogenes (e.g., miR-182, miR-205) [9,10] or tumorsuppressors (e.g., miR-7, miR-214) [11,12] in cervical cancer. How-ever, our understanding of the potential roles of miRNAs in theprognosis evaluation and treatment response of cervical cancer is

still limited. Combining miRNA expression information with clini-copathological characteristics and identifying the lead targetmRNAs are helpful in guiding the study of the functions and mech-anisms of abnormally expressed miRNAs in cervical cancer.

In our study, to screen aberrant miRNAs and predict the corre-sponding target mRNAs, high-throughput transcriptome micro-arrays were used to detect differentially expressed miRNAs andmRNAs synchronously in cervical cancer tissues compared withadjacent non-tumor tissues. From our microarray results, weinvestigated the most significantly down-regulated (>4-fold) miR-NA (Hsa-miR-145-5p, miR-145) in clinical specimens and ana-lyzed the association between miR-145 expression levels andspecific clinicopathological characteristics. Then, we combinedmicroarray analyses and bioinformatic predictions to screen can-didate target mRNAs of miR-145 effectively and accurately. Heli-case-like transcription factor (HLTF) was the best candidate targetgene, and it was found to confer radiation resistance in cervicalcancer in a recent study [13]. Moreover, we further demonstratedthat miR-145 over-expression in cervical cancer cells couldenhance radiosensitivity in vitro and in vivo, which showed aadditional anticancer function of miR-145 in cervical cancer.These findings might help establish new strategies for therapydecision-making and improving therapeutic effect of cervicalcancer.

Page 2: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

C. Ye et al. / FEBS Letters 589 (2015) 702–709 703

2. Materials and methods

2.1. Patients and samples

Human cervical tissue samples were collected from patients atthe department of Obstetrics and Gynecology at ChangzhengHospital (Shanghai, China) between 2012 and 2013. A total of 47paired cervical cancer tissues and adjacent non-tumor tissues (atP2 cm from each tumor) were obtained from patients with cervi-cal cancer (FIGO stage IB-IIB). All patients recruited in this studywere not subjected to preoperative radiotherapy and/or chemo-therapy. Besides, three normal cervical epithelial tissue sampleswere obtained from three premenopausal women (Human papillo-mavirus negative, HPV�) undergoing hysterectomy for myoma.The collection of human tissue samples was approved and super-vised by the Ethics Committee of Changzheng Hospital.

2.2. Cell culture and transfection

Human cervical cancer cell lines (HeLa, SiHa, C-33A, Caski) wereobtained from the Cell Bank of Chinese Academy of Science(Shanghai, China) and maintained as recommended by the Ameri-can Type Culture Collection (ATCC, USA). Cells were transfectedwith miR-145 mimic (RiboBio, China) or miRNA negative control(miR-NC) using the riboFECT™ CP transfection reagent (RiboBio,China) according to the manufacturer’s instructions. The final con-centration of either mimic was 50 nM in the cell culture medium.

2.3. Microarray analyses and bioinformatic predictions

Briefly, double-stranded complementary DNA (cDNA) was syn-thesized from 5 cervical cancer tissues and paired adjacent non-tumor tissues through reverse-transcription polymerase chainreactions and then hybridized to Glue Grant Human Transcriptomearrays (Affymetrix, USA) following the manufacturer’s protocol.Affymetrix� Expression Console Software (version 1.3.1) was usedfor microarray analysis. We identified differentially expressedgenes by the random variance model (RVM) t-test and false discov-ery rate (FDR) analyses, with a predefined P-value threshold of<0.05 [14]. Hierarchical clustering (Cluster 3.0) and TreeView anal-ysis (Stanford University, USA) were performed based on theresults of differentially expressed genes. TargetScan, miRdb andmiRanda combined with the differentially expressed mRNA identi-fied in the microarray were used to predict the putative targets.The microarray data discussed in this article have been submittedto the National Center for Biotechnology Information (NCBI) GeneExpression Omnibus (GEO) and are accessible through the (GEO)Series accession number GSE5594 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55940).

2.4. RNA extraction and quantitative real-time PCR

Total RNA was extracted from tissues or cells using the RNAisoPlus reagent (Takara, Japan), and cDNA was synthesized using theSuperScript� II Reverse Transcriptase kit (Invitrogen, USA). FormiRNA, RNA was reverse transcribed using a specific reverse-tran-scription primer. Quantitative real-time PCR (qRT-PCR) analyseswere performed with SYBR� Premix Ex Taq™ (Takara, Japan) usinga StepOne-plus Real-Time PCR system (ABI, USA). U6 snRNA or 18SrRNA was used as internal controls to normalize the expressionlevels of miRNAs or mRNAs, respectively. The relative expressionlevels of miRNAs or mRNAs were calculated using the 2�DDCt

method. The primers for HLTF mRNA and 18S rRNA were as fol-lows: HLTF (forward, 50-GAA ATG GAA CCA GCT GAG GCT-30;reverse, 50-TGT TCC CAG AAT GGT GGA AGT T-30); 18S (forward,

50-AAC TGC GAA TGG CTC ATT AAA TC-30; reverse, 50-TTG ATCTGA TAA ATG CAC GCA TC-30). For miR-145, we used the Bulge-Loop™ miRNA qRT-PCR Primer Set, including a specific reverse-transcription primer and miR-145 forward and reverse primers(RiboBio, China). The sequences of the primers were not providedby the manufacturer.

2.5. Vectors construction and Luciferase reporter assays

The 30 untranslated regions (30-UTR) of HLTF mRNA containingthe intact miR-145 recognition sequences were PCR-amplifiedand subcloned into the Sac I and Xba I sites of pmirGLO vector (Pro-mega, USA) for Luciferase reporter assay. The primer sequenceswere as follows: forward, 50-CGA GCT CAG TTG GGA AGT TACCTG-30; reverse, 50-GCT CTA GAG AAG GTA ATA AAC ATT TGAA-30. The pmirGLO-30 UTR with mutations in miR-145 binding siteswas synthesized by GenScript (Nanjing, China). Luciferase assayswere performed in HeLa cells. miR-145 mimic or miR-NC wasco-transfected with pmirGLO-30-UTR vector using the riboFECT™CP transfection reagent. After a 48-h incubation, firefly and Renillaluciferase activities were measured with the Dual-LuciferaseReporter system (Promega, USA) following the manufacturer’sinstructions. Firefly luciferase activity was normalized to Renillaluciferase activity for each sample.

2.6. Western blot assays

Cells were harvested in RIPA lysis buffer (Beyotime, China).Equal amounts of protein were separated by SDS–PAGE and trans-ferred onto polyvinylidene fluoride membranes (Millipore, USA).The membranes were blocked in phosphate-buffered saline/Tween-20 containing 5% non-fat milk and incubated with an anti-body against HLTF or b-actin (Santa Cruz Biotechnology, USA).Then, the membranes were incubated with HRP-labeled rabbitanti-goat IgG (KPL, USA) and detected using an Epson PerfectionV300 Photo Scanner (Epson, Japan). Quantitative analysis was per-formed using AlphaEase FC software (Alpha Innotech, USA). Proteinlevels were normalized to b-actin.

2.7. Irradiation

Cells and nude mice were exposed to 60Co-gamma ray irradia-tion at different doses (dose rate: 1 Gy/min) in the irradiation cen-ter of the Second Military Medical University (SMMU, Shanghai,China), depending on the requirements of the experiment [7].

2.8. Cell viability assay

Cells plated at 60–70% confluency in 6-well plates were trans-fected with miR-145 mimic or miR-NC and incubated for 24 h.Then, the cells were seeded in 96-well plates (1 � 104 cells perwell). After overnight incubation, the cells were irradiated at a doseof 0 Gy or 8 Gy. Then, after incubation for another 24 h, cell viabil-ity was determined using CCK-8 (Dojindo, Japan) and by measuringabsorbance at 450 nm using an ELx800™ plate reader (BioTek,USA), following the manufacturer’s instructions.

2.9. Flow cytometric analysis of cell apoptosis

Cells plated at 60–70% confluency in 6-well plates were trans-fected with miR-145 mimic or miR-NC and incubated for 24 h.Then, the cells were exposed to 0 Gy or 8 Gy irradiation. At 24 hafter irradiation, the Annexin V-FITC cell apoptosis detection kit(Beyotime, China) was used to detect apoptotic cells with a MACS-Quant� Analyzers flow cytometer (Miltenyi Biotec, Germany), fol-

Page 3: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

704 C. Ye et al. / FEBS Letters 589 (2015) 702–709

lowing the manufacturer’s instructions. The results were analyzedusing FlowJo software (Tree Star, USA).

2.10. Tumor xenograft assay

Female nude mice (BALB/C, 5 weeks old) were purchased fromShanghai Laboratory Animal Center (SLAC, China) and housedunder specific pathogen-free conditions. All the animal experi-ments were approved by the Institutional Animal Care and UseCommittee of the SMMU (Shanghai, China). To establish the subcu-taneous xenograft model, we subcutaneously injected 1 � 107

HeLa cells in 0.10 ml of phosphate-buffered saline (PBS) into theright thigh of the nude mice. Ten days after tumor cell inoculation,the cells formed palpable tumors, and the mice were divided ran-domly into three groups (four mice per group) for treatment: (1)2 nmol miR-145 agomir (cholesterol-conjugated 20-O-methyl-modified microRNA mimic, RiboBio, China) plus 4 Gy; (2) 5 nmolmiR-145 agomir plus 4 Gy; (3) 2 nmol miR-NC plus 4 Gy. For ago-mir treatment, miR-145 agomir (RiboBio, China) was directlyinjected intratumorally at a dose of 2 nmol or 5 nmol (diluted in25 lL of PBS) per mouse in the two treatment groups every 4 daysfor 6 treatments. miR-NC agomir was directly injected intratumor-ally at a dose of 2 nmol (diluted in 25 lL of PBS) per mouse in thecontrol group every 4 days for 6 treatments. All of the micereceived intratumor injection at days 12, 16, 22, 24, 28, 32, andwere exposed to 4 Gy irradiation 24 h after each injection. Thetumors were monitored with calipers, and tumor volumes were

Fig. 1. Hierarchical clustering analysis indicates the differentially expressed miRNAs in ceor green color represents expression values and indicates up- or down-regulation, respe

Fig. 2. MiR-145 expression is decreased in cervical cancer. (A) Significant down-regulationon-tumor tissues (⁄⁄⁄P < 0.001, n = 42). The relative expression levels of miR-145 were ncancer cell lines compared with normal cervical epithelial tissues.

calculated as length � (width)2/2. At day 34, the mice were sacri-ficed by cervical dislocation. The xenograft tumors were excisedand photographed.

2.11. Statistical analyses

Statistical tests for data analysis included Wilcoxon signed-ranktest, Student’s t-test, the Chi-square test, and Pearson’s coefficientcorrelation. The data are presented as the mean ± S.D. A P-value < 0.05 was considered significant. GraphPad Prism5 (Graph-Pad Software, USA) was used for the statistical analyses.

3. Results

3.1. Microarrays of aberrantly expressed miRNAs and mRNAs incervical cancer tissues

To investigate potential transcriptome changes in cervical can-cer, we performed a gene chip study in five paired cervical cancer &adjacent non-tumor tissues using the Affymetrix Human Tran-scriptome array. There were 4 up-regulated miRNAs (miR-760,922, 31-5p, 371b-3p), 8 down-regulated miRNAs (miR-145, 27b-3p, 361-5p, 361-3p, 297, 645, 24-3p, 23b-3p; Fig. 1), and 2189 dif-ferentially expressed mRNAs (1288 up-regulated, 901 down-regu-lated; Supplemental Fig. 1) detected in the microarray. Thethreshold was a fold change P1.2 with P < 0.05. Among theseresults, miR-145 was the most differentially expressed miRNA

rvical cancer tissues. A heat map shows 12 differentially expressed miRNAs. The redctively (c, cancer tissues; ad, paired adjacent non-tumor tissues).

n of miR-145 was observed in cervical cancer tissues compared with paired adjacentormalized as ln(2�DDCt). (B) The expression of miR-145 was much lower in cervical

Page 4: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

Table 1Clinical characteristics of 42 cervical cancer patients according to miR-145 expressionlevels.

Feature miR-145expression level

Chi-square P value

Low High

All cases 21 21Age, years, <45:P45 9:12 11:10 0.382 0.537Tumor size, cm, <4:P4 6:15 14:7 6.109 0.013FIGO stage, IB:IIA:IIB 4:9:8 12:5:4 6.476 0.039Degree of differentiation,

highly:moderately:poorly3:10:8 11:4:6 7.429 0.024

SCC-Ag, ng/ml, <1.5:P1.5 7:14 12:9 2.403 0.121lymph node metastasis, yes/no 8:13 11:10 0.865 0.352Hb before treatment, g/L,

<90:90–110:>1107:8:6 5:9:7 0.469 0.791

Results were considered statistically significant at P < 0.05.

C. Ye et al. / FEBS Letters 589 (2015) 702–709 705

(fold change = 4.17). Additionally, recent studies have shown thatmiR-145 is frequently down-regulated and acts as a tumor sup-pressor in various human malignancies including cervical cancer[15–18]. Therefore, we chose miR-145 for further investigation ofits significance in tumor pathogenesis and clinical treatment.

3.2. Association of miR-145 with the clinicopathological characteristicsof cervical cancer patients

Recent studies have also shown that miR-145 is down-regulated in cervical cancer; however, the relationship betweenmiR-145 expression and the clinicopathological characteristics of

Fig. 3. HLTF is directly and specifically suppressed by miR-145. (A) HLTF expression watissues. The relative expression levels of HLTF were normalized as ln(2�DDCt). (B) An invecancer tissues (n = 42, r = �0.571, P < 0.001). (C) Luciferase activities were assessed 48 h30UTR and miR-145 mimic or miR-NC in HeLa cells. miR-145 significantly repressed the ltimes. (D) Wild-type or mutant 30UTR of HLTF, indicating the interaction sites betweensignificantly reduced 48 h after miR-145 mimic or miR-NC transfection in HeLa cells, as

cervical cancer patients remains unclear [19,20]. We first exam-ined miR-145 expression levels in another 42 paired cervical can-cer & adjacent non-tumor tissues by qRT-PCR. The miR-145expression was lower in cervical cancer tissues compared withthe corresponding adjacent non-tumor tissues (Fig. 2A). Then, weevaluated the relationship between miR-145 expression and clini-copathological factors (Table 1). The median miR-145 expressionlevel in tumor tissues was used as the cutoff. We found that alower miR-145 expression was significantly more frequent in cer-vical cancer tumors with advanced stages, large tumor size andmoderate/poor differentiation. Moreover, we found that theexpression of miR-145 was much lower in cervical cancer cell linescompared with normal cervical epithelial tissues, which furthersuggested that the down-regulation of miR-145 was a commonphenomenon in cervical cancer (Fig. 2B).

3.3. HLTF is a direct target of miR-145 in cervical cancer

To determine the potential effects and molecular mechanismsof down-regulated miR-145 on the malignant behaviors of cervicalcancer cells, we focused on identifying a specific target gene ofmiR-145. We first utilized three algorithms (TargetScan, miRdband miRanda) combined with the up-regulated mRNAs from themicroarray to predict and analyze the most likely target gene. Withthis method, 5 potential target genes were identified: HLTF, ONE-CUT2, BCR, NUFIP2, and ZC3H11A. Among these results, HLTF(GenBank Accession NM_003071.3, transcript variant 1) was themost significantly up-regulated (1.96-fold) mRNA. The qRT-PCRshowed that the HLTF expression was higher in cervical cancer tis-

s assessed using qRT-PCR in cervical cancer tissues and paired adjacent non-tumorrse correlation was found between the levels of miR-145 and HLTF mRNA in cervicalafter co-transfection with pmirGLO vector containing the wild-type or mutant HLTFuciferase activity of the wild-type HLTF 30UTR. All experiments were repeated threemiR-145 and 30UTR of HLTF. (E and F) The mRNA and protein levels of HLTF weredetermined by RT-qPCR and Western blotting. ⁄P < 0.05, ⁄⁄P < 0.01.

Page 5: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

706 C. Ye et al. / FEBS Letters 589 (2015) 702–709

sues than that in corresponding adjacent non-tumor tissues(Fig. 3A). Moreover, correlation analysis showed that only HLTFexpression significantly inversely correlated with miR-145 in cer-vical cancer tissues (r = �0.571; Fig. 3B). To determine whetherHLTF is selectively regulated by miR-145, we performed luciferasereporter assays with the pmirGLO vector carrying the miR-145-complementary sequence of the wild-type or mutant HLTF 30UTRs(Fig. 3D). The two vectors were co-transfected with miR-145 mimicor miR-NC into HeLa cells. The luciferase assay results indicatedthat luciferase activity was significantly repressed by miR-145and was not affected in the mutant 30UTR group (Fig. 3C). Addition-ally, HLTF mRNA and protein levels were both inhibited in HeLacells transfected with miR-145 mimic (Fig. 3E and F). These resultsshowed that HLTF was a direct target gene of miR-145 in cervicalcancer.

3.4. Irradiation induces higher miR-145 expression levels in severalcervical cancer cell lines

Because the above results suggested that HLTF was a direct tar-get gene of miR-145 in cervical cancer cells, we attempted to deter-mine the potential role of miR-145 in the identified function andmechanism of HLTF. In recent years, several studies have demon-strated that HLTF can act as either a positive or a negative regulatorof tumor development, and it is therefore a controversial molecule[21–24]. Moreover, a recent study indicated that HLTF is an impor-tant molecule that influences the outcome of radiotherapy in cer-vical cancer [13]. Therefore, we hypothesized that miR-145 maybe associated with radiosensitivity. To test this hypothesis, we firsttreated several cervical cancer cell lines with increasing doses ofirradiation. Then, RT-qPCR analyses showed that the expressionsof miR-145 were increased after irradiation in HeLa, SiHa and Caski

Fig. 4. Radiation induces miR-145 expression in cervical cancer cells. (A–D) The radiatirespectively.⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

cells, but not in C-33A cells (Fig. 4). Because HeLa, SiHa and Caskicells contain wild-type p53, but C-33A cells harbors mutant p53.Thus, these results suggested that miR-145 might play a potentialrole in modulating the sensitivity of cervical cancer cells to radio-therapy and relying on wild-type p53.

3.5. miR-145 over-expression enhances radiation-induced cell viabilityreduction and apoptosis in cervical cancer cells in vitro

To further investigate whether miR-145 could modulate radio-sensitivity by regulating HLTF expression in vitro, we first exoge-nously up-regulated miR-145 expression in HeLa and SiHa cellsby transfecting miR-145 mimic (Fig. 5A and B). The CCK-8 assayand flow cytometric analysis showed that miR-145 mimic couldinfluence cell viability and apoptosis level alone but not veryremarkable (Fig. 5C–F, left panel). Nonetheless, radiation-inducedcell viability reduction and cell apoptosis both increased signifi-cantly by miR-145 mimic (Fig. 5C–F, right panel). These resultssuggested that miR-145 could enhance radiosensitivity of cervicalcancer cells.

3.6. Exogenous miR-145 enhances radiosensitivity of cervical cancercells in vivo

After validating that miR-145 enhanced radiosensitivity of cer-vical cancer cells in vitro, we further investigated the radiosensitiz-ing ability of miR-145 in an animal tumor model. We established aHeLa cell subcutaneous xenograft tumor model in nude mice. Themice were treated with irradiation alone (irradiation plus NC ago-mir) or in combination with the miR-145 agomir. The growthcurves and photo of xenograft tumors showed that the tumors inthe irradiation-alone group grew faster than those in the combina-

on-induced expression regulation of miR-145 in HeLa, SiHa, Caski and C-33A cells,

Page 6: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

Fig. 5. miR-145 increases radiosensitivity in cervical cancer cells. (A and B) The expression of miR-145 was detected by RT-qPCR at 48 h after miR-145 mimic or miR-NCtransfection in HeLa and SiHa cells. (C and D) At 24 h after transfection, the cells were exposed to 0 Gy or 8 Gy of 60Co-gamma ray irradiation. Then, cell viability was assessedat 24 h after irradiation. (E and F) At 24 h after transfection, the cells were irradiated with 0 Gy or 8 Gy. Then, the number of apoptotic cells was assayed by flow cytometryafter another 24 h, using PI/Annexin V-FITC double-staining. Representative graphs are shown above (early apoptosis, lower right area; late apoptosis, upper right area).⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

C. Ye et al. / FEBS Letters 589 (2015) 702–709 707

tion group. Moreover, the inhibition was stronger in the combina-tion group with the higher dose of miR-145 agomir (Fig. 6A and B).Additionally, HLTF expression decreased significantly in thecorresponding tumor tissues (Fig. 6C). Thus, these results furtherdemonstrated that miR-145 exerted a radiosensitizing effect oncervical cancer cells.

4. Discussion

Previous studies has suggested that miR-145 is down-regulatedand acts as a tumor suppressor in various human cancers [25,26].Recent articles have also reported HPV oncoproteins E6 andE7 could suppress miR-145 expression [20]. In our study, we

consistently found that miR-145 expression was remarkablydecreased in cervical cancer tissues and cell lines. And our investi-gation further discovered that decreased miR-145 was significantlyassociated with clinicopathological features, including advancedcancer stages, large tumor size and moderate/poor differentiation.

Identifying the lead specific target mRNA allows us to study themain functions and molecular mechanisms of miR-145 more effec-tively. Here, microarray analyses and bioinformatics predictionsshowed that HLTF was the top candidate target of miR-145. HLTF,belongs to the SNF/SWI family, which plays roles in chromatinremodeling and facilitates trans-factor interactions with nucleo-somes [27–29]. In our study, we found that HLTF was up-regulatedin cervical cancer tissues and inversely correlated with miR-145.

Page 7: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

Fig. 6. miR-145 reduces tumor growth in vivo combined with irradiation. (A) Growth curves of HeLa cell subcutaneous xenograft tumors treated with miR-145 agomir ormiR-NC agomir and irradiation. (B) Mice of different treatment groups were euthanized at days 34 after inoculation, and tumors were excised and photographed. (C) HLTFexpressions in xenograft tumors of different treatment groups. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

708 C. Ye et al. / FEBS Letters 589 (2015) 702–709

Furthermore, luciferase reporter assay confirmed that miR-145could selectively down-regulate HLTF by targeting the 30UTR ofHLTF.

Although several studies have identified HLTF as a tumor sup-pressor gene in digestive tract cancers [30,22], a recent articledemonstrated that HLTF confers radio-resistance in cervical cancerby enhancing DNA damage repair capacity of cancer cells [13]. Thisfinding provided us a valuable avenue for further study of the pos-sible role of miR-145 in the treatment response of cervical cancer.As expected, our results showed that miR-145 over-expressionenhanced radiosensitivity of cervical cancer in vitro and in vivothrough inhibiting cell viability and increasing radiation-inducedapoptosis probable by down-regulating HLTF. These results con-firmed the previous report of the radio-resistance role of HLTFand showed a additional anticancer function of miR-145 in cervicalcancer.

E6 and E7 are the primary HPV oncoproteins, which mainlytarget p53 and pRB, respectively [31]. It is well known that p53is a genome guardian acts as a tumor suppressor. Radiation-induced DNA damage could activate p53 and further induce cellcycle arrest and apoptosis [32]. Precious article demonstrated thatp53 transcriptionally induces the expression of miR-145 [25].Moreover, wild-type p53 is critical for the expression of miR-145 in cervical cancer cells [18]. Consistently, we found that radi-ation had no inducing effect on miR-145 expression in C-33A cellswhich harbors mutant p53. Furthermore, according to our find-ings and the conclusions above, we speculated that there mightbe a potential cascade reaction regarding miR-145 radiosensitiza-tion in cervical cancer. The process might consist of: (1) radia-tion-induced DNA damage activates p53; (2) activated p53

induces the expression of miR-145; (3) up-regulated miR-145repress HLTF, which ultimately contributes to radiosensitizingeffect in cervical cancer cell. Certainly, we need to further studyit to excavate more specific mechanism of radiosensitizing effectof miR-145.

In conclusion, our study demonstrates that decreased miR-145is associated with advanced clinicopathological characteristics ofcervical cancer, and that miR-145 contributes to radiosensitizingeffect. These findings suggest that miR-145 may be a significantpotential biomarker for prognosis evaluation and a novel radiosen-sitizing target in cervical cancer, which may help establish newstrategies for therapy decision-making and improving therapeuticeffect of cervical cancer.

5. Conflict of interest

The authors declare no conflict of interests.

Acknowledgments

This work was supported by the National Natural Science Foun-dation of China (No. 81272213, China), Natural Science Foundationof Shanghai (13ZR1414300, China) and Special Fund of the MilitaryMedical Priority Research (13CXZ006).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.febslet.2015.01.037.

Page 8: MicroRNA-145 contributes to enhancing radiosensitivity of cervical ... · MicroRNA-145 contributes to enhancing radiosensitivity of cervical cancer cells Chen Yea,1, Ning-xia Suna,1,

C. Ye et al. / FEBS Letters 589 (2015) 702–709 709

References

[1] Bartel, D.P. (2004) MicroRNAs: genomics biogenesis mechanism and function.Cell 116 (2), 281–297.

[2] Farazi, T.A., Spitzer, J.I., Morozov, P. and Tuschl, T. (2011) MiRNAs in humancancer. J. Pathol. 223 (2), 102–115.

[3] Hu, X., Macdonald, D.M., Huettner, P.C., Feng, Z., El Naqa, I.M., et al. (2009) AmiR-200 microRNA cluster as prognostic marker in advanced ovarian cancer.Gynecol. Oncol. 114 (3), 457–464.

[4] Zhao, H., Shen, J., Medico, L., Wang, D., Ambrosone, C.B., et al. (2010) A pilotstudy of circulating miRNAs as potential biomarkers of early stage breastcancer. PLoS One 5 (10), e13735.

[5] Cai, J., Yang, C., Yang, Q., Ding, H., Jia, J., et al. (2013) Deregulation of let-7e inepithelial ovarian cancer promotes the development of resistance to cisplatin.Oncogenesis 2 (10), e75.

[6] Shen, Y., Wang, P., Li, Y., Ye, F., Wang, F., et al. (2013) MiR-375 is upregulated inacquired paclitaxel resistance in cervical cancer. Br. J. Cancer.

[7] Lin, J., Liu, C., Gao, F., Mitchel, R.E.J., Zhao, L., et al. (2013) MiR-200c enhancesradiosensitivity of human breast cancer cells. J. Cell. Biochem. 114 (3), 606–615.

[8] Forouzanfar, M.H., Foreman, K.J., Delossantos, A.M., Lozano, R., Lopez, A.D.,et al. (2011) Breast and cervical cancer in 187 countries between 1980 and2010: a systematic analysis. The Lancet 378 (9801), 1461–1484.

[9] Xie, H., Zhao, Y., Caramuta, S., Larsson, C. and Lui, W.O. (2012) MiR-205expression promotes cell proliferation and migration of human cervical cancercells. PLoS One 7 (10), e46990.

[10] Tang, T., Wong, H.K., Gu, W., Yu, M.Y., To, K.F., et al. (2013) MicroRNA-182plays an onco-miRNA role in cervical cancer. Gynecol. Oncol. 129 (1), 199–208.

[11] Liu, S., Zhang, P., Chen, Z., Liu, M., Li, X., et al. (2013) MicroRNA-7downregulates XIAP expression to suppress cell growth and promoteapoptosis in cervical cancer cells. FEBS Lett. 587 (14), 2247–2253.

[12] Wang, F., Liu, M., Li, X. and Tang, H. (2013) MiR-214 reduces cell survival andenhances cisplatin-induced cytotoxicity via down-regulation of Bcl2l2 incervical cancer cells. FEBS Lett. 587 (5), 488–495.

[13] Cho, S., Cinghu, S., Yu, J.R. and Park, W.Y. (2011) Helicase-like transcriptionfactor confers radiation resistance in cervical cancer through enhancing theDNA damage repair capacity. J. Cancer Res. Clin. Oncol. 137 (4), 629–637.

[14] Wright, G.W. and Simon, R.M. (2003) A random variance model for detectionof differential gene expression in small microarray experiments.Bioinformatics 19 (18), 2448–2455.

[15] Avgeris, M., Stravodimos, K., Fragoulis, E.G. and Scorilas, A. (2013) The loss ofthe tumour-suppressor miR-145 results in the shorter disease-free survival ofprostate cancer patients. Br. J. Cancer 108 (12), 2573–2581.

[16] Gao, P., Xing, A.Y., Zhou, G.Y., Zhang, T.G., Zhang, J.P., et al. (2012) Themolecular mechanism of microRNA-145 to suppress invasion–metastasiscascade in gastric cancer. Oncogene 32 (4), 491–501.

[17] Wang, X., Tang, S., Le, S.Y., Lu, R., Rader, J.S., et al. (2008) Aberrant expression ofoncogenic and tumor-suppressive microRNAs in cervical cancer is required forcancer cell growth. PLoS One 3 (7), e2557.

[18] Shi, M., Du, L., Liu, D., Qian, L., Hu, M., et al. (2012) Glucocorticoid regulation ofa novel HPV-E6-p53-miR-145 pathway modulates invasion and therapyresistance of cervical cancer cells. J. Pathol. 228 (2), 148–157.

[19] Pereira, P.M., Marques, J.P., Soares, A.R., Carreto, L. and Santos, M.A. (2010)MicroRNA expression variability in human cervical tissues. PLoS One 5 (7),e11780.

[20] Gunasekharan, V. and Laimins, L.A. (2013) Human papillomaviruses modulatemicroRNA 145 expression to directly control genome amplification. J. Virol. 87(10), 6037–6043.

[21] Debauve, G., Capouillez, A., Belayew, A. and Saussez, S. (2008) The helicase-like transcription factor and its implication in cancer progression. Cell. Mol.Life Sci. 65 (4), 591–604.

[22] Sandhu, S., Wu, X., Nabi, Z., Rastegar, M., Kung, S., et al. (2012) Loss of HLTFfunction promotes intestinal carcinogenesis. Mol. Cancer 11 (1), 18.

[23] Capouillez, A., Decaestecker, C., Filleul, O., Chevalier, D., Coppée, F., et al.(2008) Helicase-like transcription factor exhibits increased expression andaltered intracellular distribution during tumor progression in hypopharyngealand laryngeal squamous cell carcinomas. Virchows Arch. 453 (5), 491–499.

[24] Kim, J.J., Chung, S.W., Kim, J.H., Kim, J.W., Oh, J.S., et al. (2006) Promotermethylation of helicase-like transcription factor is associated with the earlystages of gastric cancer with family history. Ann. Oncol. 17 (4), 657–662.

[25] Sachdeva, M. and Mo, Y.Y. (2010) MicroRNA-145 suppresses cell invasion andmetastasis by directly targeting mucin 1. Cancer Res. 70 (1), 378–387.

[26] Zhang, H., Pu, J., Qi, T., Qi, M., Yang, C., et al. (2012) MicroRNA-145 inhibits thegrowth, invasion, metastasis and angiogenesis of neuroblastoma cells throughtargeting hypoxia-inducible factor 2 alpha. Oncogene 33 (3), 387–397.

[27] Pazin, M.J. and Kadonaga, J.T. (1997) SWI2/SNF2 and related proteins: ATP-driven motors that disrupt protein–DNA interactions? Cell 88 (6), 737–740.

[28] Gong, X., Kaushal, S., Ceccarelli, E., Bogdanova, N., Neville, C., et al. (1997)Developmental regulation of Zbu1, a DNA-binding member of the SWI2/SNF2family. Dev. Biol. 183 (2), 166–182.

[29] Hayward-Lester, A., Hewetson, A., Beale, E.G., Oefner, P.J., Doris, P.A., et al.(1996) Cloning, characterization, and steroid-dependent posttranscriptionalprocessing of RUSH-1 alpha and beta, two uteroglobin promoter-bindingproteins. Mol. Endocrinol. 10 (11), 1335–1349.

[30] Moinova, H.R., Chen, W.D., Shen, L., Smiraglia, D., Olechnowicz, J., et al. (2002)HLTF gene silencing in human colon cancer. Proc. Natl. Acad. Sci. 99 (7), 4562–4567.

[31] Schiffman, M., Castle, P.E., Jeronimo, J., Rodriguez, A.C. and Wacholder, S.(2007) Human papillomavirus and cervical cancer. The Lancet 370 (9590),890–907.

[32] Lu, C. and El-Deiry, W.S. (2009) Targeting p53 for enhanced radio-and chemo-sensitivity. Apoptosis 14 (4), 597–606.