mirna-100 inhibits human bladder urothelial carcinogenesis ... · cancer therapeutic insights...

Cancer Therapeutic Insights

miRNA-100 Inhibits Human Bladder UrothelialCarcinogenesis by Directly Targeting mTOR

Chuanliang Xu1, Qinsong Zeng1,2, Weidong Xu1, Li Jiao1, Yanqiong Chen1, Zhensheng Zhang1,Chengyao Wu1, Taile Jin1, Anyin Pan1, Rongchao Wei1, Bo Yang1, and Yinghao Sun1

AbstractmiRNAs are involved in cancer development and progression, acting as tumor suppressors or onco-

genes. In this study, miRNA profiling was conducted on 10 paired bladder cancer tissues using 20

GeneChip miRNA Array, and 10 differentially expressed miRNAs were identified in bladder cancer and

adjacent noncancerous tissues of any disease stage/grade. After being validated on expanded cohort of

67 paired bladder cancer tissues and 10 human bladder cancer cell lines by quantitative real-time PCR

(qRT-PCR), it was found that miR-100 was downregulated most significantly in cancer tissues. Ectopic

restoration of miR-100 expression in bladder cancer cells suppressed cell proliferation and motility,

induced cell-cycle arrest in vitro, and inhibited tumorigenesis in vivo both in subcutaneous and in

intravesical passage. Bioinformatic analysis showed that the mTOR gene was a direct target of miR-100.

siRNA-mediatedmTORknockdownphenocopied the effect ofmiR-100 in bladder cancer cell lines. In addition,

the cancerous metastatic nude mouse model established on the basis of primary bladder cancer cell lines

suggested that miR-100/mTOR regulated cell motility andwas associated with tumormetastasis. Both mTOR

and p70S6K (downstream messenger) presented higher expression levels in distant metastatic foci such as in

liver and kidney metastases than in primary tumor. Taken together, miR-100 may act as a tumor suppressor

in bladder cancer, and reintroduction of this mature miRNA into tumor tissue may prove to be a therapeutic

strategy by reducing the expression of target genes. Mol Cancer Ther; 12(2); 207–19. �2012 AACR.

IntroductionIt is estimated that bladder cancer accounts for 69,250

new cases of cancer and 14,990 cancer-related deaths inthe United States in 2011 (1). It is the fourth most commoncancer in men and the eighth leading cause of death fromcancer. The ratio of men to women who develop bladdercancer is approximately 3:1 (1). The disease presents in 2different forms: non–muscle-invasive bladder cancer(NMIBC; stage Ta and T1) and muscle-invasive bladdercancer (MIBC; stage T2–4). NMIBC represents a hetero-geneous group of tumors, from completely benign non-invasive papillary tumors that rarely progress to invasivepapillary lamina propria high-grade tumors that progresstoMIBC in about 60%cases as shownby long-term follow-ups (2). Tumor grade is also an important prognostic

factor for patients with bladder cancer. Low-grade blad-der cancer is morphologically well differentiated, where-as high-grade bladder cancer is poorly differentiated andmore aggressive (3). The mechanisms of bladder cancercarcinogenesis have not been fully investigated. There-fore, a better understanding of the pathogenesis is essen-tial for the development of novel effective therapies forbladder cancer.

miRNAs are a class of small noncoding RNAmoleculesof 20 to 24 nucleotides that regulate gene expressionthrough translational repression andmRNAdegradation.So far, 1,527 human miRNAs have been registered on thebasis of the latest miRBase release 18.0 (http://www.mirbase.org/). Although their biologic functions remainlargelyunknown, recent studieshave shownthatmiRNAsplay a critical role as important regulators involved in allhallmarks of cancer (4) and that about half of the humanmiRNAs are located in cancer-associated genomic regionsand can function as tumor suppressor genes or oncogenesdepending on their targets (5). Several human miRNAshave been shown to be dysregulated in bladder cancer,including miR-125b, miR-133a, miR-143, miR-218, andmiR-221 (6–10), which contribute to the development andprogression of bladder cancer. A preliminary study (11)showed that downregulation of miR-100 was common inlow-grade bladder cancer tissues compared with normaltissues by quantitative PCR (qPCR). However, its biologicfunction remains unclear. In addition, the role of miR-100

Authors'Affiliations: 1Department ofUrology,ChanghaiHospital, SecondMilitary Medical University, Shanghai; and 2Department of Urology, Gen-eral Hospital of Guangzhou Military Command of PLA, Guangzhou, China

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

C. Xu and Q. Zeng contributed equally to this article.

Corresponding Author: Yinghao Sun, Department of Urology, ChanghaiHospital, 168 Changhai Road, Shanghai 200433, China. Phone: 86-21-81873409; Fax: 86-21-35030006; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0273

�2012 American Association for Cancer Research.

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on May 1, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0273

http://mct.aacrjournals.org/

in cancer cells seems controversial as it served as anoncogene in acute myelogenous leukemia but as a tumorsuppressor in cervical cancer (12, 13). There has been noevidence to confirm the function of miR-100 in bladdercancer.

In this study, we focused on miR-100, as our previousmiRNA array analysis on 10 pairs of human bladdercancer and adjacent noncancerous tissues showed thatmiR-100 was stably downregulated and presented highlyfold-changed expression both in NMIBC and in MIBC.After validation of the altered expression level of miR-100on 67 paired bladder cancer tissues by quantitative real-time PCR (qRT-PCR), we tested its effects on cell growth,cell-cycle distribution, colony formation, cell migration,and invasion. In addition, we also investigated the poten-tial role of miR-100 in bladder cancer tumorigenesis in 2nude mice models. Finally, we explored the underlyingmechanism of miR-100 functions in bladder cancer andvalidated its target gene, mTOR both in vitro and in vivo.Our study may hopefully provide a better understandingabout bladder cancer pathogenesis.

Materials and MethodsHuman tissues and cell lines

The tissue specimens were from 77 patients with blad-der cancer who underwent cystectomy or transurethralresection of bladder tumors between 2008 and 2010 inChanghai Hospital (Shanghai, China). The backgroundand clinicopathologic characteristics of the patients aresummarized in Supplementary Table S1. Tissue sampleswere immediately snap-frozen in liquid nitrogen. Bothtumors and adjacent normal tissues were histologicallyexamined, and all tumors were validated as urothelialcarcinoma. Our study was approved by the BioethicsCommittee of the Second Military Medical University(Shanghai, China). Written prior informed consent andapproval were given by the patients.

Bladder cancer cell lines SCaBER, BIU-87, 5637, and T24were purchased from American Type Culture Collection(ATCC).Other bladder cancer cell lines RT4, J82,HT-1376,EJ, TCCSUP, and SV40-transformed kidney cell line 293Twere kindly provided by Prof. Tianxing Lin (Sun Yat-senMemorial Hospital, Sun Yat-sen University, Guangzhou,China), who also purchased all the 6 cell lines fromATCC.Cells were passaged in our laboratory for fewer than 6months after receipt or resuscitation. Primary bladdercancer cell line 921T was established in our laboratory.It was derived under serum-free conditions as describedpreviously by Dubeau and Jones (14, 15) from a femalepatient who underwent cystectomy for a high-grade inva-sive transitional cell carcinoma of the bladder. Informedconsent was obtained from this patient.

miRNA array analysisTenpairedbladder cancer tissues (5 low-gradeNMIBCs

and5high-gradeMIBCs)were sent toCapitalBioCorp. fornoncoding RNA microarray analysis. miRNA profilingwas conducted using 20 GeneChip miRNA Array (Affy-

metrix). To respectively determine the significant differ-entially expressed miRNAs in NMIBC pairs and MIBCpairs, RVM t test analysis of microarrays was conductedusing the threshold value of �2 or �0.5. Intersection dataof the significant differentially expressed miRNAs weresubsequently obtained between the 2 pairs, log2-trans-formed and median-centered by genes using the AdjustData function of CLUSTER 3.0 software, then furtheranalyzed with hierarchical clustering with average link-age (16), and finally visualized using Java Tree View(Stanford University, Stanford, CA). The microarray plat-form and data have been submitted to the Gene Expres-sionOmnibus public database 6 at theNational Center forBiotechnology Information, following the MinimumInformation About aMicroarray Gene Experiment guide-lines. The accession numbers are GPL8786 (platform) andGSE39093 (samples; release date June 2012).

RNA extraction and quantitative real-time RT-PCRTotal RNA was extracted using TRIzol reagent (Invi-

trogen). miRNA was detected by stem-loop RT-PCR asdescribed previously (17). cDNA was synthesized withthe AccuPower RocketScript RT Premix Kit (BioNEER).The primers for RT-PCR are listed in SupplementaryTable S2.

Plasmid construction, lentivirus production, andtransduction

miR-100/control lentiviruswaspurchased fromSunbioMedical Biotechnology. Oligonucleotide transfection pre-miR-100 precursor and its negative control were pur-chased from Ambion. Cholesterol-conjugated miR-100mimics for in vivo RNA delivery and its negative controlswere from Ribobio Co. miR-100 inhibitor and mTORsiRNA (target sequence: GAACTCGCTGATCCAGATG)were synthesized by BioNEER. Oligonucleotide transfec-tion was conducted with Lipofectamine RNAiMAXreagents (Invitrogen).

Cell growth, Annexin V assays, wound-healing, andMatrigel invasion assays

Cell proliferation assay was conducted with the CellCounting Kit-8 (CCK-8; Dojindo) according to the man-ufacturer’s instruction. Cell migration and invasion assaywere conducted as described elsewhere (18). Apoptosisafter doxorubicin treatment was examined by using theAnnexin V/Propidium Iodide Detection Kit (KeyGEN)according to the manufacturer’s instructions.

Tumor formation in nude miceAll animal experimentswere undertaken in accordance

with the NIH Guide for the Care and Use of LaboratoryAnimals, with the approval of the Scientific InvestigationBoard of the SecondMilitary Medical University. Tumor-bearing female nude mice with subcutaneous passage ofEJ cells and intravesical passage of 5637 cellswereused forevaluating the antitumor effect ofmiR-100 in vivo. Bladdercancer cellswere resuspendedat a concentration of 1� 108

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Mol Cancer Ther; 12(2) February 2013 Molecular Cancer Therapeutics208




cells/mL of PBS before use. Tumor size was measured asdescribed previously (19). After 6 weeks, these mice weresacrificed and examined.For preparation of the subcutaneous model, 100 mL

suspending EJ cells infected with lenti-miR-100 or lenti-vector control were injected s.c. into the same side of theflank of the female nude mouse (4 weeks of age, eachgroup with 6 mice, n ¼ 6).In addition, another subcutaneousmodelwas prepared

using wild-type EJ cells for in vivo RNA delivery (eachgroup with 4 mice, n ¼ 4). For intratumoral injection ofcholesterol-conjugated RNA, miR-Ribo agomir-100, 2nmol RNA in 50 mL saline buffer was locally injected intoeach tumor mass at a 3-day interval for 4 weeks.The intravesical orthotopic bladder cancer model pre-

pared using the technique described by Watanabe andcolleagues (20). Briefly, after anesthesia of the femaleathymic nude mouse and transurethral insertion of a22-gauge catheter into the bladder, 100 mL 0.2% trypsinin 0.02% EDTA was infused and retained in the bladderfor 30 minutes. After trypsinization, the bladder wasrecatheterized, washed with PBS, and scratched carefullyby the catheter. Subsequently, 100 mL suspending 5637infectedwith lenti-miR-100 or vectorwas instilled into thebladder. The urethra was ligated with 6–0 nylon suture toassure that the cells were retained in the bladder. After 3hours, the suture was removed, and the bladder wasevacuated by spontaneous voiding.

Tumor metastases in nude miceWhen the animals succumbed to tumor burden and the

orthotopic bladder tumor was palpable after intravesical921T implantation (6 weeks of age, with 3mice, n¼ 3), themice were examined daily and sacrificed at 4 weeks afterimplantation or earlier if they developed signs of distress.Only obvious metastases of the lungs, liver, spleen, pan-creas, bladder, kidneys, or retroperitoneal organs wereobtained and treated for cell culturing. After validation oftheir origin by immunohistochemical staining, epithelialtumor metastasis was minced into small pieces about 0.5mm3 with scalpel blades and cultured until tumor cellswere present by the technique described previously (15).When the cells presented stable after in vitro passage, theywere harvested for intravesical implantation once again toprepare the cancerous metastatic model (6 weeks of age,with 3 mice, n ¼ 3). New metastatic tumor foci andorthotopic bladder cancer tissues of the mice were har-vested and used for evaluating the antimetastatic effectsofmiR-100 and themetastatic effects of its target gene(s) invivo.

BioinformaticsThe predicted target genes and their conserved sites of

the seed region binding with each miRNAs were inves-tigated using the TargetScan program (release 5.0, http://www.targetscan.org/). Fisher exact test was used to ana-lyze the target genes according to the Gene Ontology(GO), which is the key functional classification of NCBI

(21). False discovery rate (FDR) algorithm (22) was ap-plied to adjust the P values, using the threshold values ofFDR < 0.05. Using the KEGG database, the network ofgenes was built according to the relationship betweenmiRNAs and target genes in the database (23, 24). Tech-nical expertise for systemic bioinformatic analysis wasprovided by Novel Bioinformatics Co., Ltd.

Luciferase assayAbout 100 ng/mL of plasmid and 50 nmol/L miRNA

mimics were co-transfected into HEK 293T cells. Forty-eight hours after transfection, a Dual-Luciferase ReporterAssay System (Promega) was used to examine the effectsof miR-100 on the activity of mTOR reporter according tothe manufacturer’s protocol.

Western blot analysis and immunohistochemicalassay

Anti-mTOR mcAb and anti-p70S6K were purchasedfrom Cell Signaling Technologies. Immunohistochemicalstaining was conducted on 5-mm sections of paraffin-embedded tissue to determine the expression of AE1/AE3 (antibody from Zhongshan Goldenbridge Bio), ageneral stain for normal and neoplastic cells of epithelialorigin.

Statistical analysisThe results were presented as mean � SEM. The data

were subjected to the Student t test unless otherwisespecified (x2 test). P value of less than 0.05was consideredstatistically significant.

ResultsmiR-100 expression is decreased in human bladdercancer tissues

Twenty GeneChip miRNA array was done on 10paired bladder cancer tissues and adjacent normal tis-sues. Of the 6,703 encoded miRNAs analyzed, 20 exhib-ited significantly differential expression in low-gradeNMIBC tissues and 22 in high-grade MIBC samples(Supplementary Tables S3 and S4). Unsupervised hier-archical clustering of these significantly dysregulatedmiRNAs was able to distinguish the bladder cancersfrom corresponding normal tissues (Fig. 1A). In addi-tion, 10 miRNAs were found as an intersection set of the2 sets of significantly dysregulated miRNAs, suggestingthat they played a stable role in any tumor stage orgrade (Table 1).

To further confirm the microarray results, several tar-gets with higher fold change in the data (miR-100, -145,-182, and -210) were selected and examined in an expand-ed bladder cancer cohort consisting of 67 pairs of tumortissues and corresponding adjacent normal tissues usingqRT-PCR assay. It was found that the dysregulatedexpression level of miR-100, -145, and -182 was consistentwith the microarray data. miR-100 and -145 expressionswere stably downregulated, whereas miR-182 was upre-gulated in any tumor stage or tumor grade, judging from

Inhibitory Role of MiR-100 in Bladder Cancer

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themeanDDCt values of the 4miRNAs (Fig. 1B).However,no significant associationwas foundbetween their expres-sions in altered tumor stage or grade (P > 0.05), eventhough the mean DDCt values of both miR-100 and -145 in

invasive tumors or high-grade tumors presented slightlylower than those in non–muscle-invasive or low-gradetumors (Fig. 1B). It was interesting to find that these 2miRNAs were outstandingly downregulated in bladder

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Figure 1. miRNA expressionprofiles in bladder cancer andcorresponding noncanceroustissues. A, unsupervisedhierarchical clustering of thesignificantly dysregulated miRNAsin 5 low-grade NMIBC pairs (left)and 5 high-grade MIBC (right).Twenty exhibited significantlydifferential expression in the leftgroup, whereas 22 were exhibitedin the right. B, mean DDCt value ofmiR-100, -182, -145, and -210.miR-100 and -145 expression wasstably downregulated, whereasmiR-182 upregulated in any tumorstage or tumor grade. C, miR-100expression was examined by qRT-PCR in 67 paired bladder cancerand adjacent noncanceroustissues.

Table 1. The intersection set (10 miRNAs) between the 2 groups of significantly dysregulated miRNAs

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hsa-miR-182 86.8 25.3 3.431 0.005865 98.9 23.3 4.245 4.27E-05hsa-miR-20a 174 49.6 3.508 0.02246 176.3 59.9 2.943 8.31E-04hsa-miR-106b 125.6 35.3 3.558 0.004262 99.5 42.1 2.363 0.003637hsa-miR-210 77.5 23.3 3.326 0.045518 54.7 25.3 2.162 0.017549hsa-miR-214 68.1 170.5 0.399 0.001812 107.8 216 0.499 0.020883hsa-miR-143 311.4 726.2 0.429 0.032092 443.6 901.2 0.492 0.025808hsa-miR-145 429.5 1074.6 0.4 0.008212 531.2 1331.5 0.399 0.008439hsa-miR-125b 182.7 571.5 0.32 0.014452 228.1 619.1 0.368 0.004022hsa-miR-99a 28.4 139.5 0.204 0.011457 54.1 156.9 0.345 0.002054hsa-miR-100 82.2 233.8 0.352 0.03338 76.4 242.5 0.315 1.56E-04

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cancer. Although several studies (25, 26) clearly identifiedmiR-145 as a tumor suppressor in bladder cancer, the roleofmiR-100 in bladder cancer pathogenesiswas not furtherstudied. It was found in our study that downregulation ofmiR-100 was almost ubiquitous (53 of 67 tumors, 79.10%)and obvious in bladder cancer (Fig. 1C) either from non–muscle-invasive stage to invasive stage or from low- tohigh-grade (Fig. 1C).

miR-100 suppresses bladder cancer cell proliferationin vitro and in vivoTo determine the impact of miR-100 on bladder cancer

cell proliferation, we established EJ and 5637 transfectantsstably expressing miR-100 using lentivirus infection. Inaddition, J82 and HT-1376 were transfected with miR-100inhibitor and nonspecific control miRNA (NC). Theexpression levels of miR-100 were determined by qRT-PCR (Supplementary Fig. S1). The CCK-8 assays andcolony formation assays revealed that overexpression ofmiR-100 significantly decreased bladder cancer cell pro-liferation (P < 0.05; Fig. 2A and B), whereas RNA inter-ference–mediated silencing of miR-100 increased the cellgrowth ratio (P < 0.05; Fig. 2A).To further confirm the above findings, 3 in vivomodels

were used. Tumors in the subcutaneous nude mousemodel became palpable around 7 days in the controlgroup, whereas those in the lenti-miR100–treated groupwere palpable about 2 weeks after inoculation. All themice developed tumors at the end of the experiment (Fig.2C). The growth rate of transplanted tumors was signif-icantly different between the 2 groups in as early as 2weeks after implantation (P < 0.01). At 4 weeks afterimplantation, mice injected with lenti-vector carried larg-er burdens. The mean tumor volume of the lenti-miR100–treated group reduced markedly by more than 70% ascompared with the control (P < 0.001; Fig. 2C). No signif-icant difference in body weight was found between thelenti-miR100–treated and the control mice (P ¼ 0.056;Fig. 2C).Three mice in the lenti-miR100–treated group of the

intravesical orthotopic bladder cancer model, and allthe mice in the control group developed tumors at theend of the experiment. Tumors became palpable around3 weeks. At 5 weeks after implantation, the mean tumorvolume of the lenti-miR100–treated group reducedmarkedly by more than 60% as compared with thecontrol group (P < 0.001; Fig. 2C). No significant differ-ence in body weight was found between the 2 groups(P ¼ 0.212; Fig. 2C).Tumors in the subcutaneous model using wild-type EJ

cells for in vivo RNA delivery became palpable between 6and 8days, and all themice developed tumors. At 3weeksafter intratumoral injection of cholesterol-conjugated ago-mir-100, the mean tumor volume of the agomir-100–trea-ted group reduced significantly (P ¼ 0.028; Fig. 2D) ascompared with the control group. Taken together, ourdata indicate that miR-100 exerted an inhibitory effect onhuman bladder cancer growth.

mTOR is a direct target of miR-100All the 10 miRNAs within the intersection set were

rendered for systemic bioinformatic analysis. Totally,2,208 target genes were found, and target genes of miR-100 includedHS3ST2, SMARCA5, ZZEF1, EIF2C2, FRAP1(mTOR), and BMPR2. The high-enrichment GOs targetedby the interested miRNAs are shown in SupplementaryFig. S2. On the basis of the TargetScan and GO analysis,the potential target gene mTOR (FRAP1), whose 30-untranslated region (UTR) contains putative miR-100complementary sites, was rendered for further study (Fig.3A).

mTOR is a central element in the evolutionary con-served signaling pathway that regulates proliferation,cell growth, and survival, orchestrating signals origi-nating from growth factors, nutrients, or particularstress stimuli. The AKT and ERK/MAPK signalingpathways are 2 important modulators of mTOR activity(27). On the basis of the network of genes that we builton the basis of the KEGG database, the network of geneswithin the mTOR pathway (mTOR-miR-Network) wasfinally built (Fig. 3B). miR-100, -125b, -182, -143, -106b,-20a, -214, -143, and -145 target their correspondinggene(s) within this pathway directly or indirectly, inwhich miR-100 plays a central role among them (Fig.3B).

When conforming whether mTOR is a bona fide targetof miR-100, the relative luciferase activity of the reportercontaining wild-type mTOR 30-UTR was significantlysuppressed when miR-100 was cotransfected. In contrast,the luciferase activity of the reporter containing themutant miR-100–binding site was unaffected, indicatingthatmiR-100may suppress gene expression throughmiR-100–binding sequences at the 30-UTR of mTOR (Fig. 3A).

To find out the relationship between miR-100 expres-sion and mTOR mRNA amplification level, 10 bladdercancer cell lines were examined by qRT-PCR. Pearsoncorrelation test indicated that there was an inversecorrelation between miR-100 and the mTORmRNA level(r ¼ �0.579, P < 0.05; Fig. 3C).

Western blotting and qRT-PCR were carried out toexamine the effect of overexpression or knockdown ofmiR-100 on the mRNA and protein levels of mTOR in 2bladder cancer cell lines. mTOR mRNA and proteinexpressions were significantly decreased in EJ, 5637, andJ82 cells after transfection with pre-miR-100 (Supplemen-tary Fig. S3). Meanwhile, inhibition of endogenous miR-100 by miR-100 inhibitor resulted in upregulated expres-sion of mTOR in 5637 and J82 cells (Supplementary Fig.S3). In addition, EJ and 5637 cells were transduced withlenti-miR-100 at 3 differentmultiplicity of infection (MOI)of 10, 40, and 100 to examine mTOR expression level. Asshown inFig. 3D, ectopic expression ofmiR-100decreasedmTOR and p70S6K (p70 S6 kinase-1, its direct down-stream gene, important in cellular growth) protein levelsin a dose-dependent manner. At MOI of 100, the mTORprotein level decreased by approximately 60% to 70% ascompared with that of MOI of 10. These results indicate






that miR-100 may act as a tumor suppressor throughdownregulating the expression of mTOR in bladder can-cer cells.

Cell cycle and apoptosisEJ cellswere transfectedwith pre-miR-100, si-mTOR, or

control RNA (50 nmol/L), collected after 48 hours, and

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Figure 2. miR-100 suppresses bladder cancer cell growth in vitro and in vivo. A, top, the effect of miR-100 on cell proliferation ofmiR-100–overexpressing cells(EJ and 5637, lentivirus infected at MOI ¼ 40); bottom, inhibition of miR-100 increased the growth ratio of the bladder cancer cell lines (J82 and HT-1376,oligonucleotide transfected at 150 nmol/L). B, the effect of miR-100 on colony formation of bladder cancer cells (EJ and 5637). Representative resultsof colony formation of lenti-miR-100 (up), lenti-vector (bottom)-transfected 5637, and EJ cells. C, the effect of miR-100 on tumor formation in 2 nudemouse xenograft models. Lenti-miR-100- or vector-transfected EJ cells (1� 107) were injected s.c. into the right flank of a nude mouse (top) or intravesicallyof a nudemouse (bottom). Left, lenti-miR-100 group; right, lenti-vector group. D, the effect of intratumoral injection of cholesterol-conjugated agomir-100 ontumor growth in another nude mouse xenograft model. �, P < 0.05; ��, P < 0.01.

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stained with propidium iodide (PI) for analysis of thecell cycle by fluorescence-activated cell sorting (FACS).It was found that cells in miR-100–treated group and si-mTOR–treated group were arrested in the G0–G1 phase.This was accompanied by a corresponding decrease inthe fraction of cells in the S-phase (Fig. 4A). However,no evidence of apoptosis was seen in any of the 3groups as tested by Annexin V assays 48 hours aftercell treatment (Fig. 4B). Similarly, 5637 cells wereinfected by lenti-miR-100 or lenti-vector, and cell-cycleassay was done at 72 hours. It was also found that cellsinfected with lenti-miR-100 were arrested in the G0–G1

phase, compared with the lenti-vector–infected cells(Fig. 4C). In addition, these cells were retreated withdoxorubicin (0.15 mg/mL) 72 hours after 5637 infectionby lentivirus at MOI of 40. The results of Annexin Vassays conducted 24 and 36 hours after doxorubicintreatment showed that overexpression of miR-100induced by pretreatment with enti-miR-100 made5637 cells more sensitive to the exposure of doxorubicinsetting and enhanced the cell apoptotic effect of doxo-rubicin (Fig. 4D).

Overexpression of miR-100 reduces tumor cellmigration and invasion

The result of the scratch/wound-healing assay showedthat lenti-miR-100 treatment did reduce migration of EJand 5637 cells (Fig. 5A and B). Furthermore, miR-100upregulation, like knockdown of mTOR gene, reducedthe invasive ability of the aggressive EJ or J82 cells byapproximately 30% comparedwith control cells (P < 0.01),whereas inhibition of miR-100 could gently enhance theinvasive ability (P < 0.05; Fig. 5C and D).

The cancerousmetastatic model further suggests thefunction of miR-100 and mTOR

All the 3 mice receiving intravesical 921T implanta-tion developed bladder tumors, including one thatdeveloped bilateral kidney metastases (without uretermetastasis) 20 days after the orthotopic tumor becamepalpable (Fig. 6A). The metastatic tumor tissues of thekidneys in the mouse showed similar AE1/AE3 expres-sion when compared with the primary bladder cancertissue of the patient, suggesting that they were of epi-thelial origin (Fig. 6B). Cells from the metastases of the

Figure 3. miR-100 suppressesmTOR expression by directlytargeting its 30-UTR. A, top, putativemiR-100–binding sequence in themTOR 30-UTR. Mutation wasgenerated in the mTOR 30-UTRsequence in the complementary sitefor the seed region of miR-100 asindicated; bottom, analysis of theluciferase activity of the luciferasereporter plasmid containing eitherwild-type (wt) or mutant (mt) mTOR30-UTR. B, an mTOR-miR network.The differentially expressed miRNAsincluding miR-100, -125b, -182,-143, -106b, -20a, -214, -143, and-145 target their correspondinggene(s) within this pathway directly orindirectly. Red dots representdifferentially expressed miRNAs ofthe intersection set, blue onesrespectively represent their targetgenes, green dots represent therelated genes of the mTOR pathway.C, the expression of miR-100 andmTOR was assayed by qRT-PCR in10 bladder cancer cell lines. Pearsoncorrelation test was used to study therelationship between them. D,Western blot analysis was used tomonitor the expression level ofendogenous mTOR and p70S6K inJ82 cells after 48-hour transfectionwith pre-miR-100, miR-100 inhibitoror control RNA duplex, or in EJ and5637 cells after lenti-miR-100infection at differentMOIs. �,P<0.05;��, P < 0.01. GAPDH,glyceraldehyde-3-phosphatedehydrogenase.






right kidney were obtained and cultured, which werenamed 921T-2.

All the 3 mice receiving intravesical 921T-2 implan-tation developed tumors. Two of them also developedmetastatic tumors, including one with simultaneousliver and kidney metastases, and the other only withretroperitoneal nodal metastases (Fig. 6A). Cellsderived from these metastatic foci were cultured. It wasfound that these metastatic cells presented characteris-tic morphologic features compared with 921T cells (Fig.6A). They were usually large with abundant granularcytoplasm and/or intracytoplasmic vacuoles, suggest-ing that they had some cytoplasmic features of glandu-lar differentiation. Most nuclei were large with morethan 2 prominent nucleoli. Another distinctive feature

was the presence of spindled and/or racquet-shapedmalignant cells.

921T-2 cells and metastatic bladder cancer cells fromthe liver, kidney, and retroperitoneal lymph nodeswere collected and assayed for miR-100 and mTORexpression by qRT-PCR. As shown in Fig. 6C, miR-100 was more than 5-fold downregulated in all the 3metastatic bladder cancer cells (P < 0.01), whereasmTOR expression was upregulated correspondingly(P < 0.01), compared with that in 921T-2 cells. Espe-cially, mTOR was 8.3-fold upregulated in liver meta-static cells. In addition, Western blotting assay on thesecells suggested that protein levels of mTOR and p70S6Kwere higher both in liver and in kidney metastatic cells(Fig. 6D).

Figure 4. Cell cycle and apoptosismonitored by flow cytometry. Aand B, cell cycle and apoptosisdistribution tested 48 hours aftertransfection of EJ cells with pre-miR-100, si-mTOR, or controlRNA. C, cell-cycle distributiontested 48 hours after transfectionof 5637 cells infected with lenti-miR-100 or -vector. D, seventy-two hours after transfection of5637 cells with lentivirus at MOIof 40, cells were retreated withdoxorubicin and Annexin V assayswere conducted in 24 and 36hours. FITC, fluoresceinisothiocyanate.

Xu et al.





DiscussionRecent advances have suggested that dysregulation of

miRNAs is a common event in human cancers, includingbladder cancer. miR-182 (28, 29), -20a (30), -106b (31), -210(32), -214 (33), -143 (8, 34), -145 (25), and -125b (6) have beenreported in bladder cancer (6, 8, 25, 29) and other cancers(28, 30–34), suggesting that these miRNAs may play com-mon roles in tumorigenesis. A preliminary study (11)suggested that aberrant expression of miR-100 was almostubiquitous in low-gradebladder cancer.However, noclearinformation about the function or molecular mechanismof miR-100 in human bladder cancer has been reported.On the basis of our previous preliminary functional

tests, this study focused on miR-100, finding that miR-100 was frequently downregulated in bladder cancertissues and that miR-100 could pleiotropically inhibitcell growth and colony formation, suppress cell migra-

tion and invasion, induce G1 arrest in bladder cancercells, and suppress tumorigenesis in nude mice modelsof bladder cancer xenograft with 2 different approaches(Fig. 2), suggesting that miR-100 may act as a potentialtumor suppressor in bladder cancer. This is similar tothe findings in lung cancer (35), cervical cancer (13), andovarian cancer (36), in which miR-100 was downregu-lated, and ectopic expression of miR-100 suppressed cellproliferation and cycle. Interestingly, as predictedfrom Fig. 1, there was a potential trend that the expres-sion level of miR-100 in low-grade/stage adjacent nor-mal tissues seemed higher than that in high-grade/stage samples (P ¼ 0.112 and 0.489, respectively). Themean DDCt value of miR-100 in high-grade/stage tissuepairs presented slightly lower than that in low-grade/stage pairs (P > 0.05). Further studies with more clinicalsamples are warranted to validate the speculation thatthe microenvironment of miRNA level may be altered

Figure 5. Overexpression ofmiR-100 significantly reduces cellmigration and invasion. A and B, thescratch migration assay. Serialphotographs were obtained atindicated times posttransfection inEJ and 5637 cells. C and D, invasionrates were determined by countingthe number of cells invading throughMatrigel. Magnification, �100.�, P < 0.05; ��, P < 0.01.






and miR-100 may represent a more downward expres-sion trend in adjacent noncancerous bladder tissues ofmore aggressive bladder cancer cases, although itremains normal in histopathologic examinations.

Our in vivo data from the tumor metastasis modelestablished with primary bladder cancer cells suggestthat altered expression ofmiR-100played a role in bladdercancer cellular metastasis. Downregulated expression of

miR-100 was implicated with nodal/distant metastasis.However, recent studies (37, 38) showed thatmiR-100wasamplified and a high level of miR-100 was related tobiochemical recurrence of localized prostate cancer inpatients. These controversial results suggest that the roleof miR-100 is possibly tumor-specific and highly depen-dent on its targets in different cancer cells. Indeed, thetissue- and time-dependent expression of miRNAs may

Figure 6. The cancerous metastatic model further suggests the function of miR-100 and mTOR. A, top, one mouse developed orthotopic tumors and bilateralkidney metastases 20 days after intravesical 921T implantation. After intravesical 921T cell implantation, one mouse developed liver and kidneymetastases simultaneously and the other with retroperitoneal nodal metastases. Bottom, characteristic morphologic features of metastatic cells obtainedfrom the live and retroperitoneal nodes compared with 921T cells. B, AE1/AE3 expression detected by immunohistochemistry in the metastatic tumortissues of the kidneys and the primary bladder cancer tissue (921T-origined) of the patient. C, miR-100 expression downregulated in all the 3 metastaticbladder cancer cells (P < 0.01), whereas mTOR upregulated correspondingly (P < 0.01) compared with that in 921T-2 cells. D, Western blotting assaysuggested that protein levels of mTOR and p70S6K were higher both in liver and in kidney metastatic cells. Magnification, �200.

Xu et al.





influence protein translation during distinct cellular pro-cesses, and the aberrant expression of their target genesmay affect different biologic pathways with diverse func-tions (39).It was reported that mutation or overexpression of

FGFR3 gene occurs in approximately 80% patients withNMIBC. It can activate the Ras kinase signaling pathway,leading to increased proliferation and motility. In con-trast, MIBCs mainly contain mutations in TP53 gene.Thus, a well-established 2-pathway model of bladdercancer has been established (40). In recent years, mTOR,a key downstream effector of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, has been recog-nized to play a crucial role in controlling cancer cellgrowth (41). AKT and mTOR function as "master switch"proteins in cancer cells tomodulatemetabolism, cell cycle,and apoptosis (42). Inhibition of mTOR results in a widevariety of effects on normal andmalignant cells, includinginduction of apoptosis and inhibition of cell-cycle pro-gression, cell growth, angiogenesis, endothelial cell pro-liferation, and protein translation. As a hot research point,the mTOR signaling pathway has also been increasinglyimplicated in bladder cancer tumorigenesis (43, 44).On the basis of the systemic bioinformatic analysis, the

mTOR-miR network that we built showed that miR-100played a central role in this pathway (Fig. 3B). Then, weshowed that miR-100 inhibited mTOR expression in adose-dependent manner and confirmed that mTOR wasa direct target of miR-100 in bladder cancer cells (Fig. 3).Knockdown of mTOR induced cell growth inhibition andG1 phase arrest without stimulating apoptosis, whichwassimilar to the phenotypes induced bymiR-100 restoration(Fig. 4). These results suggest that the inhibitory effect ofmiR-100 on bladder cancer cell growth was mediated byrepressing mTOR expression and was attributed to theregulation of cell-cycle progression, particularly at the G1

to S-transition. This may occur by blocking the down-stream messenger, p70S6K, and preventing the transla-tion of some key mRNAs required for cell-cycle progres-sion (45). Our results expand the horizon of previouspreclinical studies on the role of the mTOR signalingpathway in bladder cancer (43, 44). Particularly, rapamy-cin and its derivative everolimus (RAD001) have beennoted to inhibit bladder cancer cell proliferation andinduce G0–G1 cell-cycle arrest without apoptosis in blad-der cancer cell lines. The efficacy of mTOR inhibition onbladder cancer proliferation and tumor growth has alsobeen shown previously in mouse xenografts (44).Increasing evidence has shed new insights into the

role of mTOR in the regulation of cell motility (46). It isknown that mTOR functions as 2 complexes, mTORC1and mTORC2. Earlier studies have shown the participa-

tion of mTORC2 signaling in cytoskeletal events andcell movement (47). Recent studies (48, 49) have furthershown that rapamycin-sensitive mTORC1 also plays acrucial role in cell regulation by phosphorylating 4E-BP1(eukaryotic initiation factor 4E–binding protein 1) andp70S6K. Both mTORC1-mediated p70S6K and 4E-BP1pathways are involved in the regulation of cell motility.P70S6K regulates cell motility, probably by regulatingphosphorylation of the focal adhesion proteins (48). How-ever, little is known about how 4E-BP1 pathway regulatescellmotility.We found that knockdownofmTORreducedthe cell-invasive ability similar to the phenotype as shownbymiR-100 overexpression (Fig. 5). In addition, data fromthe cancerous metastatic model further suggest that miR-100 andmTOR regulated cellmotility andwere associatedwith tumormetastasis. BothmTORandp70S6Kpresentedhigher expression levels in distant metastatic foci such asin liver and kidney metastases.

Taken together, this study identified miR-100 as agrowth-suppressive miRNA in human bladder cancer, atleast partly, through repression of mTOR. Our data pro-vide further evidence that miRNAs play a pivotal role inbladder cancerdevelopment andprogression.AsmiR-100is downregulated in bladder cancer, reintroduction of thismature miRNA into tumor tissue may prove to be atherapeutic strategy by reducing the expression of targetgenes. Although miRNA-based therapeutics is still intheir infancy, our findings on miR-100 are encouragingand suggest that this miRNA could be a potential targetfor the treatment of bladder cancer in future.

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

Authors' ContributionsConception and design: C. Xu, Q. Zeng, Y. SunDevelopment of methodology: C. Xu, Q. Zeng, Y. Cheng, C. Wu, Y. SunAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Q. Zeng, W. Xu, T. Jin, A. Pan, R. WeiAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): Q. Zeng, L. Jiao, Z. Zhang, A. Pan, R. WeiWriting, review, and/or revision of the manuscript: Q. Zeng, L. Jiao, C.Wu, B. YangAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Cheng, Y. SunStudy supervision: C. Xu, Y. Sun

Grant SupportThis work was supported by the National Natural Science Foundation

of China (grant no. 81172425 to C. Xu).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMarch 15, 2012; revisedNovember 9, 2012; acceptedDecember3, 2012; published OnlineFirst December 27, 2012.

References1. Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the

impact of eliminating socioeconomic and racial disparities on prema-ture cancer deaths. CA Cancer J Clin 2011;61:212–36.

2. CooksonMS,HerrHW,ZhangZF,SolowayS,Sogani PC, FairWR. Thetreated natural history of high risk superficial bladder cancer: 15-yearoutcome. J Urol 1997;158:62–7.






3. Pollard C, Smith SC, Theodorescu D. Molecular genesis of non-muscle-invasive urothelial carcinoma (NMIUC). Expert Rev Mol Med2010;12:e10.

4. Croce CM. Causes and consequences of microRNA dysregulation incancer. Nat Rev Genet 2009;10:704–14.

5. Ryan BM, Robles AI, Harris CC. Genetic variation in microRNA net-works: the implications for cancer research. Nat Rev Cancer 2010;10:389–402.

6. Huang L, Luo J, Cai Q, Pan Q, Zeng H, Guo Z, et al. MicroRNA-125bsuppresses the development of bladder cancer by targetingE2F3. Int JCancer 2011;128:1758–69.

7. Uchida Y, Chiyomaru T, Enokida H, Kawakami K, Tatarano S, Kawa-hara K, et al. MiR-133a induces apoptosis through direct regulation ofGSTP1 in bladder cancer cell lines. Urol Oncol. 2011 Mar 9. [Epubahead of print].

8. Noguchi S, Mori T, Hoshino Y, Maruo K, Yamada N, Kitade Y, et al.MicroRNA-143 functions as a tumor suppressor in human bladdercancer T24 cells. Cancer Lett 2011;307:211–20.

9. Lu Q, Lu C, Zhou GP, Zhang W, Xiao H, Wang XR. MicroRNA-221silencing predisposed human bladder cancer cells to undergo apo-ptosis induced by TRAIL. Urol Oncol 2010;28:635–41.

10. Tatarano S, Chiyomaru T, Kawakami K, Enokida H, Yoshino H, HidakaH, et al. miR-218 on the genomic loss region of chromosome 4p15.31functions as a tumor suppressor in bladder cancer. Int J Oncol2011;39:13–21.

11. Catto JW, Miah S, Owen HC, Bryant H, Myers K, Dudziec E, et al.Distinct microRNA alterations characterize high- and low-grade blad-der cancer. Cancer Res 2009;69:8472–81.

12. Zheng YS, ZhangH, Zhang XJ, Feng DD, Luo XQ, ZengCW, et al. MiR-100 regulates cell differentiation and survival by targeting RBSP3, aphosphatase-like tumor suppressor in acute myeloid leukemia. Onco-gene 2012;31:80–92.

13. Li BH, Zhou JS, Ye F, Cheng XD, Zhou CY, Lu WG, et al. ReducedmiR-100 expression in cervical cancer and precursors and itscarcinogenic effect through targeting PLK1 protein. Eur J Cancer2011;47:2166–74.

14. Dubeau L, Jones PA. Growth of normal and neoplastic urothelium andresponse to epidermal growth factor in a defined serum-free medium.Cancer Res 1987;47:2107–12.

15. Dubeau L, Jones PA. A new in vivo model to study invasion andmetastasis of human bladder carcinoma. Cancer Res 1987;47:6660–5.

16. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis anddisplay of genome-wide expressionpatterns. ProcNatl AcadSci 1998;95:14863–68.

17. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, et al.Real-time quantification of microRNAs by stem-loop RT-PCR. NucleicAcids Res 2005;33:e179.

18. Shi W, Gerster K, Alajez NM, Tsang J, Waldron L, Pintilie M, et al.MicroRNA-301 mediates proliferation and invasion in human breastcancer. Cancer Res 2011;15:2926–37.

19. Hou J, Lin L, Zhou W, Wang Z, Ding G, Dong Q, et al. Identification ofmiRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. CancerCell 2011;19:232–43.

20. Watanabe T, Shinohara N, Sazawa A, Harabayashi T, Ogiso Y, Koya-nagi T, et al. An improved intravesical model using human bladdercancer cell lines to optimize gene and other therapies. Cancer GeneTher 2000;7:1575–80.

21. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al.Gene ontology: tool for the unification of biology. The Gene OntologyConsortium. Nat Genet 2000;25:25–29.

22. Reiner A, Yekutieli D, Benjamini Y. Identifying differentially expressedgenes using false discovery rate controlling procedures. Bioinformat-ics 2003;19:368–75.

23. Li C, Li H. Network-constrained regularization and variable selectionfor analysis of genomic data. Bioinformatics 2008;24:1175–82.

24. Zhang JD, Wiemann S. KEGG graph: a graph approach to KEGGPATHWAY in R and bioconductor. Bioinformatics 2009;25:1470–71.

25. Ostenfeld MS, Bramsen JB, Lamy P, Villadsen SB, Fristrup N,Sørensen KD, et al. miR-145 induces caspase-dependent and -inde-pendent cell death in urothelial cancer cell lines with targeting of anexpression signature present in Ta bladder tumors. Oncogene 2010;29:1073–84.

26. Chiyomaru T, Enokida H, Tatarano S, Kawahara K, Uchida Y,Nishiyama K, et al. miR-145 and miR-133a function as tumour sup-pressors and directly regulate FSCN1 expression in bladder cancer. BrJ Cancer 2010;102:883–91.

27. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth andmetabolism. Cell 2006;124:471–484.

28. Sun Y, Fang R, Li C, Li L, Li F, Ye X, et al. Hsa-mir-182 suppresses lungtumorigenesis through down regulation of RGS17 expression in vitro.Biochem Biophys Res Commun 2010;396:501–7.

29. Han Y, Chen J, Zhao X, Liang C, Wang Y, Sun L, et al. MicroRNAexpression signatures of bladder cancer revealedbydeepsequencing.PLoS One 2011;6:e18286.

30. Huang G, Nishimoto K, Zhou Z, Hughes D, Kleinerman ES. miR-20aencoded by the miR-17–92 cluster increases the metastatic potentialof osteosarcoma cells by regulating Fas expression. Cancer Res2012;72:908–16.

31. Li B, Shi XB, Nori D, Chao CK, Chen AM, Valicenti R, et al. Down-regulation of microRNA 106b is involved in p21-mediated cell cyclearrest in response to radiation in prostate cancer cells. Prostate2011;71:567–74

32. Roth�e F, Ignatiadis M, Chaboteaux C, Haibe-Kains B, Kheddoumi N,Majjaj S, et al. GlobalmicroRNAexpression profiling identifiesmiR-210associatedwith tumor proliferation, invasionandpoor clinical outcomein breast cancer. PLoS ONE 2011;6:e20980.

33. Derfoul A, Juan AH, Difilippantonio MJ, Palanisamy N, Ried T,Sartorelli V. Decreased microRNA-214 levels in breast cancer cellscoincides with increased cell proliferation, invasion and accumu-lation of the Polycomb Ezh2 methyltransferase. Carcinogenesis2011;32:1607–14.

34. Liu L, Yu X, Guo X, Tian Z, Su M, Long Y, et al. MiR-143 is down-regulated in cervical cancer and promotes apoptosis and inhibitstumor formation by targeting Bcl-2. Mol Med Report 2012;5:753–60.

35. Feng B, Wang R, Chen LB. MiR-100 resensitizes docetaxel-resistanthuman lung adenocarcinoma cells (SPC-A1) to docetaxel by targetingPlk1. Cancer Lett 2012;317:184–91

36. Nagaraja AK, Creighton CJ, Yu Z, Zhu H, Gunaratne PH, Reid JG, et al.A link between mir-100 and FRAP1/mTOR in clear cell ovarian cancer.Mol Endocrinol 2010;24:447–63.

37. Leite KR, Tomiyama A, Reis ST, Sousa-Canavez JM, Sa~nudo A,Dall'Oglio MF, et al. MicroRNA-100 expression is independently relat-ed to biochemical recurrence of prostate cancer. J Urol 2011;185:1118–22.

38. Leite KR, Sousa-Canavez JM, Reis ST, Tomiyama AH, Camara-LopesLH, Sa~nudo A, et al. Change in expression of miR-let7c, miR-100, andmiR-218 from high grade localized prostate cancer to metastasis. UrolOncol 2011;29:265–9.

39. Ambros V. The functions of animal microRNAs. Nature 2004;431:350–5.

40. Goebell PJ, Knowles MA. Bladder cancer or bladder cancers? Genet-ically distinct malignant conditions of the urothelium. Urol Oncol2010;28:409–28.

41. Guertin DA, Sabatini DM. Defining the role of mTOR in cancer. CancerCell 2007;12:9–22.

42. Castedo M, Ferri KF, Kroemer G. Mammalian target of rapamycin(mTOR): pro- and anti-apoptotic. Cell Death Differ 2002;9:99–100.

43. Hansel DE, Platt E, Orloff M, Harwalker J, Sethu S, Hicks JL, et al.Mammalian target of rapamycin (mTOR) regulates cellular proliferationand tumor growth in urothelial carcinoma. Am J Pathol 2010;176:3062–72.

44. Mansure JJ, Nassim R, Chevalier S, Rocha J, Scarlata E, KassoufW. Inhibition of mammalian target of rapamycin as a therapeuticstrategy in the management of bladder cancer. Cancer Biol Ther2009;8:2339–47.

45. Rowinsky EK. Targeting the molecular target of rapamycin (mTOR).Curr Opin Oncol 2004;16:564–75.

Xu et al.





46. Zhou H, Huang S. mTOR signaling in cancer cell motility andtumor metastasis. Crit Rev Eukaryot Gene Expr 2010;20:1–16.

47. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, et al.Mammalian TOR complex 2 controls the actin cytoskeleton and israpamycin insensitive. Nat Cell Biol 2004;6:1122–28.

48. Berven LA, Willard FS, Crouch MF. Role of the p70(S6K) pathway inregulating the actin cytoskeleton and cell migration. Exp Cell Res2004;296:183–95.

49. Liu L, Chen L, Chung J, Huang S. Rapamycin inhibits F-actin reorga-nization and phosphorylation of focal adhesion proteins. Oncogene2008;27:4998–5010.






2013;12:207-219. Published OnlineFirst December 27, 2012.Mol Cancer Ther Chuanliang Xu, Qinsong Zeng, Weidong Xu, et al. Directly Targeting mTORmiRNA-100 Inhibits Human Bladder Urothelial Carcinogenesis by

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