Identiﬁcation of miR-133b and RB1CC1 as Independent Predictors for Biochemical Recurrence and Potential Therapeutic … · Digital images were captured using Nikon DS-Ri1 ECLIPSE

Human Cancer Biology

Identification of miR-133b and RB1CC1 as IndependentPredictors for Biochemical Recurrence and PotentialTherapeutic Targets for Prostate Cancer

Xia Li1, XuechaoWan1, Hongbing Chen3, Shu Yang1, Yiyang Liu1, WenjuanMo1, Delong Meng1, Wenting Du1,Yan Huang1, Hai Wu1, Jingqiang Wang1, Tao Li2, and Yao Li1

AbstractObjective: We aimed to investigate the contribution of microRNA-133b (miR-133b) in prostate cancer

cell proliferation, cell cycle, and apoptosis. We also examined expression of miR-133b in prostate cancer

tissues, and evaluated the prognostic significance of miR-133b, as well as its target gene RB1CC1 in patients

with prostate cancer after radical prostatectomy.

Experimental Design: miR-133b mimics (miR-133bm) and anti–miR-133b were transfected into

LNCaP and PC-3 cells. CCK-8 was used to look at cell proliferation, flow cytometric analysis was carried

out to study cell cycle, and apoptosis was determined by caspase-3 activity. miR-133b expression was

assessed by real-time reverse transcription PCR and in situ hybridization in prostatic cell lines and 178

prostate tissue samples, respectively. The protein level of RB1CC1 was examined by Western blot and

immunohistochemistry in prostatic cell lines and prostate tissue samples, respectively.

Results: Overexpression of miR-133b in LNCaP cells boosted cell proliferation and cell-cycle progres-

sion, but inhibited apoptosis; in contrast, miR-133bm promoted cell apoptosis, but suppressed cell

proliferation and cell-cycle progression in PC-3 cells. In LNCaP cells, silencing of RB1CC1, a target of

miR-133b, inhibited cell apoptosis, and promoted cell-cycle progression. Moreover, miR-133b expression

was significantly inversely correlated with RB1CC1 expression in prostate cancer tissues. Multivariate Cox

analysis indicated thatmiR-133b and RB1CC1might be two independent prognostic factors of biochemical

recurrence.

Conclusions: miR-133b might enhance tumor-promoting properties in less aggressive LNCaP cells,

whereas this miRmay act as a tumor suppressor inmore aggressive PC-3 cells. miR-133b and RB1CC1were

independent prognostic indicators for prostate cancer. Clin Cancer Res; 20(9); 2312–25. �2014 AACR.

IntroductionProstate cancer is the most frequently diagnosed cancer

and the second leading cause of cancer-related deaths in themale population of the United States (1). It has beenreported that nearly 217,730 new cases of prostate cancerwere diagnosed in the United States in 2010, among which

92% are present as local and low-stage cases, as indicated byprostate-specific antigen (PSA) testing (2). To date, radicalprostatectomy (RP) is the standard treatment for localizedprostate cancer (3). However, about 25% to 50% post-RPpatients on long-term follow-up have biochemical recur-rence (BCR; ref. 4). This is generally the earliest indicator ofthe recurrence. Therefore, the early risk detection of BCRwith sensitive methods and specific tumor markers mayeffectively improve the prognosis of patients with prostatecancer.

Androgen has been reported to play a pivotal role inandrogen pathways and prostate carcinogenesis (5). Andro-gen receptor (AR) regulates androgen-dependent gene tran-scription (6). Many AR-regulated genes are crucial regula-tors of prostate development and maintenance (7). Recent-ly, emerging evidence suggests the potential involvement ofaltered regulation of microRNAs (miRNA) in AR signalingand the pathogenesis of prostate cancer (8–11).

miRNAs are short, endogenous RNAs in cells, promotingtranslational repression and/or destabilizing targetmRNAs,to optimize protein levels in numerous biologic processes(12).MicroRNA-133b (miR-133b), located at chromosome

Authors' Affiliations: 1State Key Laboratory of Genetic Engineering,Institute of Genetics, School of Life Sciences, Fudan University; 2Depart-ment of Urology, Tongji Hospital, Tongji University School of Medicine,Shanghai; and 3Department ofUrology,Hefei First People'sHospital, Hefei,Anhui, People's Republic of China

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Yao Li, Room 604 Science Building, State KeyLaboratory of Genetic Engineering, School of Life Sciences, Fudan Uni-versity, 220 Handan Road, Shanghai 200433, People's Republic of China.Phone: 86-21-65642047; Fax: 86-21-65642502; E-mail:[email protected]; and Tao Li, Department of Urology, Tongji Hospital,Tongji University School ofMedicine, Shanghai 200433, People's Republicof China. Phone: 86-21-66111533; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1588

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(9) May 1, 20142312

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Published OnlineFirst March 7, 2014; DOI: 10.1158/1078-0432.CCR-13-1588

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6p12.2, is downregulated in several solid cancers (13–15).miR-133b has been reported to function as an oncomiR ortumor suppressor in different types of cancers (16–18).However, few studies have focused on the role of miR-133b in prostate cancer, especially the significance of miR-133b expression in clinical prognostic.RB1-inducible coiled-coil 1 (RB1CC1), identified as a

potential regulator of RB1 gene (19), is found in chromo-some 8q11, a region containing several loci of putativetumor suppressors. RB1CC1 has been reported as a criticalsignaling node that coordinately regulates cell growth,cell proliferation, apoptosis, and cell spreading/migration(20). It is also associated with various types of humancancer, especially primary breast cancer (21). However,the role of RB1CC1 in prostate cancer and the significanceof RB1CC1 expression in clinical diagnosis were rarelyreported.In this study, we investigated miR-133b expression in

cell lines and clinical samples, and analyzed the potentialassociation between miR-133b and various clinicopath-ologic factors of patients with prostate cancer. We iden-tified that miR-133b displayed tumor-promoting proper-ties in LNCaP cells, whereas acted as a tumor suppressorin PC-3 cells. miR-133b and RB1CC1, a target of miR-133b, were independent prognostic indicators for pros-tate cancer.

Materials and MethodsPatientsA total of 135 specimens of patients with prostate cancer,

18 patients with prostatic intraepithelial neoplasia (PIN),and 25 normal prostate tissue samples were included in this

study. The normal prostate tissue samples were derivedfrom patients with bladder cancer after total cystectomy;PIN and prostate cancer samples were derived frompatients with prostate cancer after RP and regional lymphnode dissection in Tongji Hospital, subsidiary of TongjiUniversity (Shanghai, China), between January 2001 andNovember 2012. None of the patients received any pre-operation treatment. Cases were classified according toWorld Health Organization criteria and staged accordingto tumor, node, metastasis classification and the Gleasongrading system. The Research Ethics Committee of TongjiHospital approved this protocol, and verbal consentswere obtained from all patients. BCR was defined as apostoperative serum PSA concentration � 0. 2 ng/mL(22).

Cell culture and androgen treatmentThe prostate cancer cell lines LNCaP, 22Rv1, PC-3, and

DU145, and the immortalized noncancerous prostaticstromal cell line WPMY-1 were obtained from Cell Bankof Chinese Academy of Sciences (Shanghai, China) wherethey were authenticated by mycoplasma detection, DNAfingerprinting, isozyme detection, and cell vitality detec-tion. The four prostate cancer cell lines were maintainedin RPMI 1640 medium, and WPMY-1 cells were culturedin Dulbecco’s Modified Eagle’s medium. The androgentreatment assay was performed as described previously(23). According to previous reports (24–26), 10 nmol/Ldihydrotestosterone (DHT) was found to be the optimalconcentration to stimulate LNCaP cell proliferation.

miRNA mimics and inhibitor molecule treatment andRNA interference

Synthetic miR-133b mimic (miR-133bm) and its scram-bled control, control miRNA (miR-NC), siRNA againstRB1CC1 gene, and its negative control (an siRNA lackinghomology to the genome) were fromGenePharma (Shang-hai, China), and used at 50 nmol/L concentration. Anti–miR-133b (si133b; catalog number IH-300709-06-0005)and its scrambled control, control anti-miRNA (siNC; cat-alog number IN-001005-01-05) were from Thermo FisherScientific, and at 100 nmol/L concentration. All sequencesof synthetic oligonucleotides are listed in SupplementaryTable S3.

Transient transfectionTransient transfection was performed as described previ-

ously (27). The Opti-MEM medium and Lipofectamine2000 were both from Life Technologies.

Real-time quantitative reverse transcription PCRReal-time quantitative reverse transcription PCR (qRT-

PCR) for mRNAs was performed as described in (23). FormiRNA quantification, cDNA was synthesized from totalRNA with the MiScript Reverse Transcription Kit (Qia-gen). Specific primers for mature miR-133b were fromQiagen (catalog number MS00031430). The primers usedfor qPCR were listed in Supplementary Table S4.

Translational RelevanceThe identification of novel molecular prognostic bio-

markers and therapeutic targets for prostate cancer isfundamental toward improvement of patient outcome.MicroRNAs represent a new class of biomarkers anddrugtargets. Here, we identify and validate the dual role ofmicroRNA-133b (miR-133b) as amediator of aggressivephenotype in LNCaP (androgen-dependent) cells and asa tumor suppressor in PC-3 (androgen-independent)cells. We have also identified miR-133b target, RB1CC1gene, which functions as a tumor suppressor in LNCaPcells. We performed in situ hybridization and immuno-histochemistry on tissue microarrays from 178 prostatetissue specimens, showing thatmiR-133bwas associatedwith poor prognosis, whereas RB1CC1 was associatedwith favorable prognosis. The expression of miR-133bwas negatively correlated with RB1CC1 protein levels.Taken together, these results suggested that miR-133bmight play crucial roles in cellular processes of prostatecancer progression, and that miR-133b and RB1CC1 areindependent biomarkers of prognosis in prostate cancer.

The Role of miR-133b in Prostate Cancer

www.aacrjournals.org Clin Cancer Res; 20(9) May 1, 2014 2313




Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) was performed

as described in (23). With minor modification, LNCaPcells were treated with vehicle, DHT (100 nmol/L), anti-androgen Casodex (10 mmol/L), or a combination of DHTandantiandrogen for 4hoursbefore harvesting. Theprimersof androgen response element (ARE) for qPCR were listedin Supplementary Tables S5 and S6. KLK3 (PSA) enhancerwas used as the positive control (28), whereas XBP-1 pro-moter andaDNAregion adjacent tomiR-133bgenewithouta putative ARE served as the negative controls (29).

Expression constructsExpression construct was performed as described previ-

ously (30). Briefly, the wild-type 30UTR (untranslatedregion) of RB1CC1was amplified by PCR from LNCaP cell,and was inserted downstream of the stop codon of lucifer-ase. Two pairs of primers, Mut-1 and Mut-2, were used togenerate a 5 nucleotides substitution mutation of the puta-tive miR-133b binding sites with the KOD-Plus-Mutagen-esis Kit (Toyobo). The primers are listed in SupplementaryTable S7.

Dual luciferase assayLNCaP cells were cotransfected in 24-well plates with 20

pmol miR-133bm or miR-NC, together with 0.8-mg fireflyluciferase report construct containing the wild-type ormutant RB1CC1-30-UTR and 8-ng control vector pRL-TK(Promega). Forty eight hours after transfection, firefly andrenilla luciferase activities were measured with GloMax 96Microplate Luminometer (Promega).

Western blotWestern blot was performed as described previously (23)

with antibodies against RB1CC1 (1:1,000; Sigma-Aldrich),b-actin (1:4,000; Sigma-Aldrich), and EGFR (1:1,000;Proteintech).

Cell proliferation assayCell proliferation was performed as described in (27).

Briefly, cells were transfectedwithmiR-133bmor anti–miR-133b, and their negative control, respectively, and exam-ined at 0, 24, 48, and 72 hours. At each time point, CCK-8reagent was added into cells and incubated for 2 hours. Theabsorbance was measured at 450 nm using a multimodemicroplate reader (BioTek).

Cell-cycle assayCell cycle was performed as described in (31). Briefly,

cells were incubated with propidium iodide (10 mg/mL) for15minutes in the dark. The fractions of viable cells in G1, S,and G2 phases of cell cycle were measured with a FACStarflow cytometer (Becton-Dickinson).

Apoptosis assaysmiR-133bm, miR-NC, anti–miR-133b, or anti–miR-NC

were transfected as described above. Forty eight hours aftertransfection, cells were collected and assayed with the FITC

Annexin V Apoptosis Detection Kit (Becton-Dickinson) ona FACScalibur flow cytometer following the manufacturer’sinstructions.

Intracellular caspase and caspase-3 activitiesIntracellular caspase and caspase-3 assays were per-

formed as described previously (32). Activities of the totalcaspase and caspase-3 were assayed with the CaspGLOWFluorescein Caspase Staining Kit and CaspGLOW Fluores-cein Caspase-3 Staining kit (BioVision), respectively, anddetected by FACScalibur flow cytometer.

Tissue microarray constructionTissue microarray (TMA) construction was performed as

described previously (33). Two replicate tumor samples(1mmindiameter)were taken from thedonor tissue blocksin a highly representative fashion and arrayed into a recip-ient paraffin block (35 mm � 622 mm � 65 mm) with atissue microarrayer (Beecher Instrument Inc.), as describedby Kononen (34).

Locked nucleic acid in situ hybridization andimmunohistochemistry of TMA

Locked nucleic acid in situ hybridization (LNA-ISH)was performed as described previously (33) on a set ofprostate cancer TMA slides using LNA probes for miR-133b(Exiqon; sequence: /5DigN/TAGCTGGTTGAAGGGGAC-CAAA/3Dig_N/). Expression of RB1CC1 was detected inimmunohistochemistry assays performed as described pre-viously (35), with polyclonal antibody for RB1CC1 (1:100;Proteintech).

Image acquisition and managementDigital images were captured using Nikon DS-Ri1

ECLIPSE Series (Nikon) with a 10� objective. TMAimages were managed using NIS Element D4.00 software(Nikon).

Statistical analysisThe BCR-free survival time was defined as the time from

the date of surgery to that of BCR, and the follow-up datawere updated until November 2012. The differential expres-sions of RB1CC1 between groups were analyzed using c2

analysis. The probability of survival was estimated accord-ing to the Kaplan–Meier method. With the log-rank test, weanalyzed the differences between the survivals of differentpatients groups. To analyze the correlation between miR-133b and RB1CC1 expression and clinicopathologic char-acteristics, the t test, c2 analysis, Mann–WhitneyU test, andFisher exact test were used according to the test condition. ACox proportional hazards model was performed to estab-lish independent factor(s) for patients’ survival. Statisticalsignificance was defined as P < 0.05, and all tests were two-tailed. The statistical analysis was performed based on theSPSS software, version 15.0 (SPSS Inc.).

For experiments in cell lines, mean and SDs of individualgroups (n � 3) were calculated. P values were assessed byperforming the two-tailed Student t test.

Li et al.

Clin Cancer Res; 20(9) May 1, 2014 Clinical Cancer Research2314




ResultsAndrogen induces the expression of miR-133bWe have previously analyzed miRNA and mRNA expres-

sion in LNCaP cells treatedwith 10nmol/LDHT at differenttime points (23). cDNAs reverse transcribed from totalRNAs at each timepointwere hybridized to Illumina SentrixHumanWG-6_V2 expression BeadChip arrays (for mRNA)orMicroRNA Expression Profiling Panels (for miRNA). Theraw data have been uploaded to the Gene ExpressionOmnibus public repository (http://www.ncbi.nlm.nih.gov/geo/; Gene Expression Omnibus series numberGSE21245). Data preprocessing, filtering criteria, and sta-tistical analysis were as described in (23). Of the 137differentially expressed miRNAs, miR-133b was found tobeandrogen-responsive in early stage afterDHT stimulation(20 minutes, 8 hours), and had higher ARE enrichmentaccording to genomatix database prediction (36). Further,miRDB database was applied to predict the sequence-basedmiRNA targets (37), which were also androgen-responsivegenes, and GenMAPP for the pathway enrichment of miR-133b. As a result, miR-133b was valued with significantpathway enrichment in key cellular processes, such asregulation of cell size, cell growth, and cell cycle (P <0.05, data not shown).Our microarray data analysis identified miR-133b to be

upregulated before 4 hours, with a peak at 1 hour after DHTstimulation (Fig. 1A). Further validation of miR-133bexpression was performed by qRT-PCR in LNCaP cellsunder the same experimental conditions. The expressionof miR-133b was increased compared with the vehiclecontrol (Fig. 1B). The expressions of two androgen-respon-sive genes PSA and KLK2 were used as positive controls(Supplementary Fig. S1A and S1B).

Differential expression of miR-133b in prostatic celllinesNext, we evaluated the basal expression of miR-133b in

noncancerous prostatic cells WPMY-1 and four prostatecancer cell lines LNCaP, 22Rv1, PC-3, and DU145. LNCaPis androgen-dependent prostate cancer (ADPC) cell line;22Rv1 is weakly androgen-dependent; PC-3 is androgen-independent prostate cancer (AIPC) cell line without ARexpression; andDU145 is AIPC cell linewith AR expression.We observed that the less aggressive LNCaP cells had theleast expression of miR-133b, whereas the more aggressivePC-3 cells showed the highest expression, which was 395.5-fold over that of LNCaP cells (Fig. 1C). The finding raisedthe possibility that differentially expressedmiR-133bmightcontribute to the progression of prostate cancer. In thisstudy,we focused onmiR-133b and investigated its involve-ment in prostate cancer.

Verification of the binding of AR to miR-133bAs we have confirmed the expression of miR-133b is

upregulated by androgen, we wondered whether AR couldbind to the DNA region of the miR-133b locus. We usedGenomatix database (36) to predict AREs within the

upstream and downstream 10 kb from pre–miR-133b’stranscription start site. Seven AREs, formed 5 clusters(named A–E), were detected in the proximal DNA regionsof miR-133b (Fig. 1D). By ChIP–qPCR, we found that ARrecruitment onto six of seven AREs was induced significant-ly after 100 nmol/L DHT stimulation for 4 hours comparedwith the vehicle (Fig. 1E). Treatment with solo-Casodex or acombination of DHT and Casodex significantly reduced ARrecruitment to levels comparable with vehicle. The PSAenhancer and XBP-1 promoter served as positive controland negative control for AR binding, respectively (Fig. 1F).Taken together, our results suggested that androgen–ARsignaling mediated the regulation of miR-133b in LNCaPcells.

miR-133b influenced cell proliferation and cell-cyclestatus of prostate cancer

Because miR-133b was directly regulated by AR, weanalyzed the role of miR-133b on various cellular processesof prostate cancer. First, we evaluated the effect ofmiR-133bon cell proliferation of LNCaP, 22Rv1, and PC-3 cells. Thesecells were transfected with miR-133bm or anti–miR-133b(si133b). We observed that when cells were treated withmiR-133bm, cell proliferation was significantly increasedcompared with miR-NC in LNCaP and 22Rv1 cells (Fig. 2Aand C), whereas anti–miR-133b partially decreased cellproliferation compared with anti–miR-NC in these two celllines (Fig. 2B andD). In contrast, in PC-3 cells, miR-133bminhibited cell proliferation, whereas si133b boosted cellproliferation (Fig. 2E and F). The transfection efficiency ofthese assays was analyzed by qRT-PCR (Supplementary Fig.S1C–S1E).

As cell-cycle distribution is a parameter reflecting thegrowth of cells, we assessed the function of miR-133b oncell-cycle profile of LNCaP and PC-3 cells. Overexpressionof miR-133b in LNCaP cells induced an increase in S phaseand a decrease in G1 phase compared with miR-NC, where-as knockdown of miR-133b resulted in a reduction in Sphase and an increase in G1 phase (Fig. 2G and Supple-mentary Fig. S2A). However, in PC-3 cells, miR-133bmcaused an increase in G1 phase and a decrease in S phase,whereas si133b decreased the proportion of cells in G1

phase and increased the proportion in S phase (Fig. 2Hand Supplementary Fig. S2C). The transfection efficiency ofthese assays is shown in Supplementary Fig. S2B and S2D.Collectively, miR-133b boosted cell proliferation andgrowth of ADPC cells LNCaP by promoting cell-cycle pro-gression, but suppressed cell proliferation and growth ofAIPC cells PC-3.

miR-133b affected cell apoptosis of prostate cancerWe further explored the role of miR-133b on cell apo-

ptosis of prostate cancer. LNCaP and PC-3 cells were treatedwith miR-133bm, si133b, or their negative control, respec-tively, and followed by flow cytometry analyses. In LNCaPcells, miR-133bm boosted the fraction of living cells anddecreased that of late apoptotic cells compared with themiR-NC, whereas si133b increased both the fraction of






early apoptotic and late apoptotic cells (Fig. 3A and Sup-plementary Fig. S3A).On the other hand, in PC-3 cells,miR-133bm reduced the fraction of living cells and boosted thatof late apoptotic cells, but si133b increased the proportionof living cells and decreased that of late apoptotic cells(Fig. 3B and Supplementary Fig. S3C). The transfectionefficiency of these assays is shown in Supplementary Fig.S3B and S3D.

To clarify the mechanism by which miR-133b affectedcell apoptosis of prostate cancer, we detected the activity oftotal caspase and caspase-3 in LNCaP and PC-3 cells. InLNCaP cells,miR-133bm significantly reduced the fractionsof activated caspase (Fig. 3C and Supplementary Fig. S4A)and caspase-3 (Fig. 3E and Supplementary Fig. S4E), ascompared with miR-NC; however, si133b boosted theproportion of activated caspase. In PC-3 cells, miR-133bm

Figure 1. miR-133b expression to response androgen and the basal expression in different prostatic cell lines. A, time course profile of miR-133bexpression in response to 10 nmol/L DHT stimulation in LNCaP cells. The Y-axis referred to normalized signal intensity of the miRNA microarray. B,LNCaP cells were treated with 10 nmol/L DHT or ethanol as vehicle at different time points as indicated. Total RNA was prepared as described inMaterials and Methods and analyzed for miR-133b expression by qRT-PCR. All expression levels were normalized to vehicle control at eachtime point, each experiment was performed in triplicate, and values are mean � SD. C, qRT-PCR analysis of miR-133b expression level in differentprostatic cell lines as indicated. The data were expressed as fold change of miR-133b expression in each cell line versus LNCaP cells. D,schematic diagram of miR-133b's ARE sites at the 50 and 30 regions. ARE site is indicated by arrows. E and F, occupancy of AR on candidate AREs ofmiR-133b, PSA enhancer, and XBP-1 promoter with antiandrogen treatment. LNCaP cells were treated with vehicle, 10 mmol/L antiandrogen Casodex(CDX), 100 nmol/L DHT, or a combination of 100 nmol/L DHT and 10 mmol/L Casodex (indicated as DHTþCasodex) for 4 hours before harvestingfor ChIP assays. Chromatin samples were immunoprecipitated with anti-AR antibody, and bound DNA was quantified as described in Materialsand Methods. E, AR occupancy on the candidate AREs of miR-133b. A DNA region adjacent to miR-133b gene (9982–9895-bp upstream frompre–miR-133b transcription start site) that does not contain a putative ARE served as negative control (indicated as NC). F, AR occupancy on the PSAenhancer (positive control) and XBP-1 promoter (negative control). Values, expressed as percentages of input DNA, are the mean � SD of at least threeindependent experiments. Significance is defined as P < 0.05 (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

Li et al.





significantly increased the fractions of activated caspase(Fig. 3D and Supplementary Fig. S4C) and caspase-3(Fig. 3F and Supplementary Fig. S4G), whereas si133b

decreased the proportion of activated caspase. The trans-fection efficiency is shown in Supplementary Fig. S4B, S4D,S4F, and S4H, respectively. Taken together, we found that

Figure 2. miR-133b played critical roles in cell proliferation and cell cycle in prostate cancer cells. A to F, cell proliferation analysis. Cellswere seeded into a 96-wellplate at 5,000 cells/well and examined at 0, 24, 48, and 72 hours after transfection. Each experiment was performed in sextuplicate (n ¼ 6). miR-133boverexpression boosted cell proliferation in LNCaP (A) and 22Rv1 (C), and inhibited proliferation in PC-3 cells (E); knockdown of miR-133b suppressed cellproliferation in LNCaP (B) and 22Rv1 (D), and promoted proliferation in PC-3 cells (F). G and H, cell-cycle analysis. Cells were transfected with miR-133bm,miR-NC, si133b, or siNC for 48 hours, and stained with propidium iodide (PI), and then subjected to cell-cycle analysis. G, miR-133b overexpression caused adecrease inG1phaseandan increase inSphase inLNCaPcells; knockdownofmiR-133bcausedan increase inG1phaseandadecrease inSphase.H,miR-133boverexpressioncausedan increase inG1phaseandadecrease inSphase inPC-3cells; anti–miR-133bcausedadecrease inG1phaseandan increase inSphase.






Figure3. miR-133b involved inprostatecancercell apoptosis.Cellswere transfectedwithmiR-133bmormiR-NC, si133borsiNC for48hours,and thensubjectedto cell apoptosis (stained with PI and FITC-Annexin V), caspase (stained with FITC-VAD-FMK), and caspase-3 (stained with FITC-DEVD-FMK) analyses. A, inLNCaP cells, miR-133b overexpression caused a decrease in late apoptotic cells and an increase in living cells; knockdown of miR-133b increased theproportions of both early and late apoptotic cells. B, in PC-3 cells, miR-133b overexpression caused a decrease in living cells and an increase in late apoptoticcells; anti–miR-133b caused an increase in living cells, and a decrease in late apoptotic cells. C, in LNCaP cells, miR-133b overexpression suppressed theactivitiesof intracellularcaspase; knockdownofmiR-133bpromoted theactivityof intracellularcaspase.D, inPC-3cells,miR-133boverexpressionpromoted theactivities of intracellular caspase; anti–miR-133b suppressed the activity of intracellular caspase. E, miR-133b overexpression suppressed the activities ofcaspase-3 in LNCaP cells. F, miR-133b overexpression boosted the activities of caspase-3 in PC-3 cells. Each experiment was performed in triplicate.

Li et al.





miR-133b functioned as an oncomiR, which promotedcell proliferation and suppressed apoptosis of ADPC cellsLNCaP; however, this miR acted as a tumor suppressor byinhibiting cell proliferation and promoting apoptosis ofAIPC cells PC-3.

RB1CC1 is a target of miR-133bTo identify potential target of miR-133b, we conducted

a computational screen for genes with complementarysites of miR-133b in their 30UTR using open-access soft-ware [TargetScan (www.targetscan.org), PicTar (http://pictar.mdc-berlin.de/), and miRDB (www.mirdb.org)].We have performed expression profile in LNCaP cellsand found several androgen-responsive miRNAs andmRNAs. To precisely evaluate the actual regulation ofeach predicted miRNA–mRNA pair, the modulation score(MS) was proposed (23). For each predicted miRNA–mRNA pair whose both members were androgen respon-sive, MS was calculated and the P value for each MS wasevaluated. Subsequently, four mRNAs were identified ascandidate targets of miR-133b: RB1CC1, PTPRK, SESN1,and CPNE3. qRT-PCR analysis in LNCaP cells indicatedthat the expression of RB1CC1 was the most significantlyaffected by overexpression or knockdown of miR-133bamong these putative targets (Supplementary Fig. S5A).Further, we focused on RB1CC1, which was reported tobe a tumor suppressor and activate the expression of RB1in breast cancer (38), but its role in prostate cancer isunclear.To confirm RB1CC1 as the direct target of miR-133b,

we performed luciferase assays in LNCaP cells. There isan miR-133b eight-nucleotide seed match region at posi-tion 252–259 of the RB1CC1 30-UTR (Fig. 4A). Cotrans-fection of LNCaP cells with the wild-type 30-UTR-reporterand miR-133bm showed significantly decreased lucifer-ase activity as compared with miR-NC (Fig. 4B). Inter-action between the binding site in RB1CC1 30-UTR andmiR-133b was sequence-specific, because mutation of theseed sequence restored luciferase activity to values com-parable with the empty vector (Fig. 4B). These resultsdenoted that the 30 UTR of RB1CC1 transcript was atarget of miR-133b.

Ectopic expression of miR-133b affected endogenousRB1CC1After we transiently transfected LNCaP, 22Rv1, and PC-3

cells with miR-133m, si133b, or their negative controls,respectively, miR-133bmwas shown to decrease the mRNAand protein level of RB1CC1 relative to miR-NC, whereassi133b increased the mRNA and protein level of RB1CC1compared with siNC (Fig. 4C and D and SupplementaryFig. S5C).To further address the physiologic relationship between

endogenousmiR-133bandRB1CC1 in prostate cancer cells,RB1CC1 protein level and miR-133b expression were eval-uated by Western blot (Fig. 4E) and qRT-PCR (Fig. 4F) infour prostatic cell lines, respectively. A significantly inversecorrelation betweenRB1CC1protein andmiR-133b expres-

sion was found, with a Pearson correlation coefficient of�0.831 (P < 0.05; Fig. 4G).

Because androgen induced expression of miR-133b, andmiR-133b downregulated RB1CC1, we went further toevaluate changes of RB1CC1mRNA and protein levels after10 nmol/LDHT stimulation for 24 hours in LNCaP cells. Asa result, androgen significantly decreased mRNA and pro-tein level of RB1CC1, relative to vehicle control (Supple-mentary Fig. S5B). Collectively, these results suggested thatexpression of RB1CC1 and miR-133b was closely linked inprostate cancer cells, supporting RB1CC1 as an endogenoustarget of miR-133b.

Knockdown of RB1CC1 expression boosted cell-cycleprogression and inhibited cell apoptosis

To evaluate how RB1CC1 functioned in cell cycle andcell apoptosis processes of prostate cancer, we usedRB1CC1 siRNA to knock down the expression of RB1CC1.As shown in LNCaP cells, knockdown of RB1CC1 signif-icantly decreased the proportion of cells in G1 phase andincreased that of cells in S phase (Supplementary Fig. S6Aand S6B), similar to that observed in the overexpressionof miR-133b (Fig. 2G).

Cell apoptosis analysis revealed that knockdown ofRB1CC1 boosted the fraction of living cells and reducedthat of late apoptotic cells compared with negative control,similar to that observed in the overexpression of miR-133b(Supplementary Fig. S6C and S6D). The transfection effi-ciency of RB1CC1 siRNA is shown in Supplementary Fig.S6E.

Taken together, we found that RB1CC1, a target of miR-133b, functioned as a tumor suppressor by inducing G1–S-phase arrest and promoting cell apoptosis of LNCaP cells.

Expression ofmiR-133b and RB1CC1 in prostate tissuesamples

To assess the clinical relevance of these observations incell lines, we evaluated the expression of miR-133b andRB1CC1 on prostate cancer TMA. LNA-ISH was per-formed to detect miR-133b expression for 178 prostatetissue specimens mounted on a set of prostate cancer TMAslides. ISH score of miR-133b was estimated by scanningeach TMA spot. Figure 5A and B showed representativemiR-133b staining in TMAs. Among the 135 prostatecancer samples, 92 cases (68.1%) showed positive expres-sion of miR-133b, compared with 14 of 18 PIN (77.8%)samples and 7 of 25 normal prostate tissues (28.0%),respectively, which was statistically significant (c2 ¼16.432; P < 0.0001).

Immunohistochemistry assay was conducted to analyzeRB1CC1 protein level in 178 prostate tissue specimens.Figure 5C and D showed the representative staining ofRB1CC1 protein in TMAs. Among the 135 prostate cancersamples, 54 cases (40.0%) showed positive expression ofRB1CC1 protein, compared with 6 (33.3%) of 18 PINsamples and 20 (80.0%) of 25 normal prostate tissues,respectively, which was statistically significant (c2 ¼14.730; P < 0.001).






Figure 4. miR-133b directly targeted RB1CC1. A, the binding site of miR-133b to the 30-UTR of RB1CC1. Vertical arrows represent the mutated bases in theRB1CC1-30UTRmutant reporterconstructs.B,LNCaPcellswereseeded intoa24-wellplate,RB1CC1 reporterconstruct (wild-typeormutant)or theempty reportervectorwas cotransfectedwithmiR-133bmandpRL-TK, or cotransfectedwithmiR-NC andpRL-TK.miR-133b overexpression decreased the luciferase activity ofRB1CC1-30UTR wild-type (RB1CC1), but not that of RB1CC1-30UTR-mutant (MUT) or the empty reporter vector (empty). (Continued on the following page.)

Li et al.





Correlation between expression of miR-133b andRB1CC1 protein in prostate cancer samples and theirrelationship to clinical variablesTo explore the correlation between miR-133b and

RB1CC1 expression, we performed both the Pearson andSpearman rank correlation coefficient analyses. A signif-icantly inverse correlation between RB1CC1 protein andmiR-133b expression was found, with a correlation coef-ficient of �0.180 (P ¼ 0.036; Supplementary Tables S8

and S9). We also examined the relationship between theexpression of miR-133b, RB1CC1, and various clinico-pathologic features in patients with prostate cancer afterRP. As shown in Supplementary Table S1, positive miR-133b expression was significantly associated with patho-logic stage (P ¼ 0.012) and BCR (P ¼ 0.038), whereaspositive RB1CC1 protein expression was significantlyassociated with preoperation PSA (P ¼ 0.021), Gleasonscore (P ¼ 0.012), and BCR (P ¼ 0.024).

Figure 5. miR-133bandRB1CC1 aspotential prognostic markers forprostate cancer. A to D, miR-133band RB1CC1 expression inprostate cancer samples. A, therepresentative positive expressionof miR-133b was mainly detectedin prostatic epithelial cells. B, therepresentative negative staining ofmiR-133b in prostatic epithelialcells. C, the representative positivestaining of RB1CC1 on tissuearrays. D, RB1CC1 staining wasnegative in prostate cancersamples. The magnification is100�. E and F, the Kaplan–Meieranalysis showed BCR-free survivalof patients with prostate cancerpatients. E, miR-133b. F, RB1CC1.

(Continued.) CandD,qRT-PCRandWesternblottingshowed theendogenousexpressionofRB1CC1 in LNCaPcells (C)andPC-3cells (D) followedby transfectionwithmiR-133bm,miR-NC,si133b,orsiNC.The results revealed thatoverexpressionofmiR-133brepressed theendogenousexpressionofRB1CC1comparedwiththatofmiR-NCcontrol, andanti–miR-133bcouldpartially restore theexpressionofRB1CC1. E,Westernblotanalysis ofRB1CC1protein levels in differentprostaticcell lines as indicated. Image quantification was performed using Quantity One software. Data were normalized using b-actin protein level. Fold changewas presented as relative RB1CC1 protein level of each cell line versus LNCaP cells. F, qRT-PCR analysis of miR-133b basal expression in different prostatic celllines as indicated. Data were normalized using RNU6B expression. Fold change was presented as relative miR-133b expression (log10) of each cell line versusLNCaP cells. G, correlation between RB1CC1 protein levels andmiR-133b expression showed a high degree of reverse correlation (R¼�0.831;P¼ 0.023). Eachexperiment was performed in triplicate. Values are mean � SD.






miR-133bandRB1CC1wereprognostic factors forBCRafter RP

To further examine whether there was significantly dif-ferent outcome between patients with negative and positivemiR-133b expression, the risk of BCR was evaluated usingthe Kaplan–Meier method, and a statistically significantseparation of survival curves was observed (Fig. 5E). Fur-thermore, the 5-year BCR-free survival probability rateand mean survival time were estimated (SupplementaryTable S2). We also estimated the probability of survival inpatientswithnegative andpositive RB1CC1 expression, andthe mean BCR-free time and 5-year BCR-free survival rates.A statistically significant separation of survival curves wasobserved (Fig. 5F).

Meanwhile, the Cox proportional hazards model wasused to assess independent predictors of BCR-free survival,the factors including preoperation PSA, Gleason score,surgical margin, lymph node metastasis, capsular invasion,pathologic stage, and the expression of miR-133b andRB1CC1 protein. As a result, miR-133b was identified asan independent prognostic factor of outcome for patientswith prostate cancer who underwent RP [HR (95% CI),1.775 (1.013–3.108), P ¼ 0.045], as well RB1CC1 [HR(95% CI), 0.534 (0.316–0.902), P ¼ 0.019], preoperationPSA [HR (95%CI), 1.009 (1.002–1.016), P¼0.012], tumormargins [HR (95% CI), 0.470 (0.224–0.984), P ¼ 0.045],andGleason score [HR (95%CI), 2.242 (1.215–4.137), P¼0.010; Table 1]. Thus, our findings suggested thatmiR-133bexpression and RB1CC1 protein level could independentlypredict the risk of BCR for patients with prostate cancer afterRP.

DiscussionThe miRNA expression profiles of many solid malignan-

cies have been reported (33). Compared with traditionalmRNA and proteinmarkers,miRNA expression patterns aremore reliable and sensitive to changes in cell biology.mRNA and its translated protein level are not often pro-portional, an important reason for which is the regulatoryinfluences of epigeneticmechanisms, including thosemedi-ated by miRNAs (39).

Specifically, miRNAs can act as oncomiRs or tumorsuppressors in a multitude of cancers (40). As referred to

miR-133b, in cervical carcinoma cells, it enhanced cellproliferation and colony formation, suggesting that it couldact as an oncomiR (16). On the other side, miR-133b wasreported to be a tumor suppressor in tongue squamous cellcarcinoma (SCC; ref. 13), esophageal SCC (14), and colo-rectal cancer cells (15).

Our previous study had shown that miR-133b signifi-cantly boosted AR-induced cell viability of LNCaP cells, andmiR-133b was required for mediating AR signaling toprostate cancer cell viability and survival (23). In this study,we found that miR-133b stimulated proliferation of LNCaPand 22Rv1 cells, whereas inhibited apoptosis of LNCaPcells. Therefore, miR-133b played an oncogenic role inADPC cells LNCaP and 22Rv1. On the other hand, twoprevious articles demonstrated tumor suppressive functionsof miR-133b in AIPC cells PC-3 and DU145 (41, 42). Bothof these studies showed that miR-133b contributed greatlyto the suppression of proliferation in PC-3 cells. Consistentwith their reports, we observed thatmiR-133b inhibited cellproliferation and cell-cycle progression, whereas promotedapoptosis of PC-3 cells. It has been shown that the samemiRNA could have opposing roles in different cellularsettings (43). Collectively, these data indicated that miR-133b might display tumor-suppressing properties in AIPCcells PC-3, but act as an oncomiR in ADPC cells LNCaP and22Rv1.

At present, we asked which factor affects or regulatesthe cellular abundance of miR-133b in prostate cancercells. We have found that androgen stimulation inducedan enhanced abundance of miR-133b in LNCaP cells.Furthermore, we observed that the AR is recruited tothe ARE region of miR-133b in the presence of DHT.These findings suggest that androgen–AR signalingmay mediate the regulation of miR-133b in AR-positiveprostate cancer cells. AR may associate with additionalregions or may require other factor(s) to associate effi-ciently with the miR promoter. Further characterizationof androgen-dependent and androgen-independent reg-ulations of miRNA expression is currently in progress inour group.

A previous study demonstrated that EGFR was a target ofmiR-133b in prostate cancer (42). In line with their results,we found that miR-133b downregulated the expression of

Table 1. Multivariate Cox regression analysis of BCR–free survival for all patients with prostate cancer afterRP

Variables P HR for recurrence 95% CI

miR-133b expression 0.045 1.775 1.013–3.108RB1CC1 expression 0.019 0.534 0.316–0.902Preoperation PSA 0.012 1.009 1.002–1.016Tumor margins 0.045 0.470 0.224–0.984Gleason score 0.010 2.242 1.215–4.137

NOTE: Significant contributing factors in multivariate Cox regression analysis weremiR-133b expression, RB1CC1 expression, tumormargins, preoperation PSA, and Gleason score.


Li et al.




EGFR in LNCaP andPC-3 cells (Supplementary Fig.S5DandS5E).In 2012, Patron and colleagues performedwhole-genome

microarray expression analysis after transfection of miR-133bm inHeLa cells andmatched the list of downregulatedgenes with miRecords (41). However, RB1CC1 was not onthe list, because HeLa cells are cervical cancer cells, and ourdata were obtained in prostate cancer cells. As we know, thesamemiRNA can target differentmRNAs indifferent cellularcontext (43). Further experiments will be necessary to clarifywhether RB1CC1 is the target of miR-133b in HeLa cells.Notably, recent studies suggested that RB1CC1 could

represent a potential diagnosis and drug target for a broadrange of cancers. Nuclear expression of RB1CC1was shownto predict a better clinical outcome in salivary gland cancers(44). In breast cancer,RB1CC1was reported to predict long-term survival together with RB1 and p53 (45). On the basisof our results, we speculated thatRB1CC1might function asa tumor suppressor, which was opposite to the oncomiRrole of miR-133b in ADPC cells LNCaP, yet further study isneeded to fully illustrate the function of RB1CC1 in AIPCcells.To our knowledge, this is the first study showing the

significance ofmiR-133b andRB1CC1 in relation to clinicaldata on patients with prostate cancer. We found that theexpression of miR-133b and RB1CC1 protein was inverselycorrelated. Because none of the patients involved in ourstudy received any preoperation treatment, the clinicalsamples can be regarded as androgen dependent. Hence,our findings in patients’ specimens confirmed the results weobtained in ADPC cells LNCaP, which indicated an onco-miR role of miR-133b and a tumor suppressor function ofRB1CC1. Finally, multivariate Cox hazard regression anal-ysis revealed that miR-133b and RB1CC1were identified asindependent prognostic factors of outcome for patientswith prostate cancer after RP.Despite the combination of increasingly refined surgical

techniques and reduced incidence of surgical complica-tions, the variable disease course leads to eventual recur-rence of about one third of patients following RP (46).Pound and colleagues reported that distant or local recur-rence of prostate cancer has not occurred in males withoutBCR (47). Therefore, to achieve long-term disease-freesurvival for patients with prostate cancer, BCR risk shouldbe assessed. Recent investigations have tried to apply theimprovedunderstandingof tumor cell biology to determineadditional tumor characteristics as potential prognosticfactors. Such prognostically important factors might helpdetermine the optimal treatment strategy based on thebiologic potential of individual tumor (48). On the basis

of this study, patients with prostate cancer with low expres-sion of miR-133b or high protein level of RB1CC1 shouldundergo regular monitoring of serum PSA and clinicalsymptoms. In contrast, patients with high expression ofmiR-133b or low protein level of RB1CC1 could benefitfrom obtaining additional clinical data including ultra-sound-guided biopsy, computed tomography, magneticresonance imaging, and bone scans.

In conclusion, our results ascertained miR-133b as adirect target of AR in LNCaP cells. miR-133b acted as apotent antiapoptotic and progrowth molecule with onco-miRs properties in ADPC cells LNCaP, but functioned as apotential tumor suppressor in AIPC cells PC-3. miR-133btargeted and downregulated RB1CC1 in prostate cancercells. The expression of miR-133b and RB1CC1 protein isinversely correlated, and they can be used to predict the riskof BCR in patients with prostate cancer after RP. Althoughevaluations ofmiR-133b expression andRB1CC1protein inlarger populations are still needed, our results indicated thatmiR-133b and RB1CC1 might be promising candidates asmolecular prognostic biomarkers and therapeutic targetsfor prostate cancer.

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

Authors' ContributionsConception and design: X. Li, X. Wan, Y. LiDevelopment of methodology: X. Li, H. Chen, H. WuAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): X. Li, X. Wan, H. Chen, Y. Liu, W. Du, H. WuAnalysis and interpretation of data (e.g., statistical analysis, biostatist-ics, computational analysis):X. Li, X.Wan, S. Yang, Y. Liu,W.Mo,D.Meng,W. Du, J. Wang, T. LiWriting, review, and/or revision of the manuscript: X. Li, Y. Huang,J. Wang, T. LiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): X. Li, X. Wan, W. Mo, W. Du, Y. LiStudy supervision: Y. Li

AcknowledgmentsThe authors thank Drs. Aditya Dutta, Shazia Irshad, Chee Wai Chua, and

Songyan Han for the comments, thoughtful suggestions, and revision ofthe English language in the article. They also thank Chuan Li for helpfuldiscussions.

Grant SupportThis work is supported by grants 31071142 and 31171246 from the

National Natural Science Foundation of China.The costs of publicationof this articlewere defrayed inpart by thepayment

of page charges. This articlemust therefore be herebymarked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 17, 2013; revised January 26, 2014; accepted February 25,2014; published OnlineFirst March 7, 2014.

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2014;20:2312-2325. Published OnlineFirst March 7, 2014.Clin Cancer Res Xia Li, Xuechao Wan, Hongbing Chen, et al. Prostate Cancerfor Biochemical Recurrence and Potential Therapeutic Targets for Identification of miR-133b and RB1CC1 as Independent Predictors

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