Molecular and Cellular Pathobiology

MicroRNA-708 Induces Apoptosis and SuppressesTumorigenicity in Renal Cancer Cells

Sharanjot Saini, Soichiro Yamamura, Shahana Majid, Varahram Shahryari, Hiroshi Hirata,Yuichiro Tanaka, and Rajvir Dahiya

AbstractCancer pathogenesis is restricted by stresses that compromise cell division and survival. In this study, we

identify miR-708, a little studied member of a set of microRNAs that have been implicated in stress control, as animportant tumor suppressor in renal cell carcinoma (RCC). miR-708 expression was attenuated widely in humanRCC specimens. Restoration of miR-708 expression in RCC cell lines decreased cell growth, clonability, invasion,and migration and elicited a dramatic increase in apoptosis. Moreover, intratumoral delivery ofmiR-708 was sufficient to trigger in vivo regression of established tumors in murine xenograft models ofhuman RCC. Investigation of the targets of miR-708 identified the inhibitor of apoptosis protein survivin asimportant. siRNA-mediated knockdown of survivin partially phenocopied miR-708 overexpression suggestingthat the proapoptotic role of miR-708 may be mediated primarily through survivin regulation. Additionally, weidentified the E-cadherin regulators ZEB2 and BMI1 as likely miR-708 targets. Taken together, our findingsdefine a major tumor suppressive role for miR-708, which may offer an attractive new target for prognostic andtherapeutic intervention in RCC. Cancer Res; 71(19); 6208–19. �2011 AACR.

Introduction

The incidence and mortality rates of renal cell carcinomas(RCC) has increased in recent years with approximately 50,000new cases and 12,000 deaths in 2010. Approximately 30% oflocalized RCC cases develop metastatic recurrence (1) withvery poor prognosis because of the refractory nature of RCC tothe current treatment regimens. Therefore, there has beenmuch interest in the identification of biomarkers for RCCwhich could lead to development of better prognostic, diag-nostic, and therapeutic interventions for the disease.

MicroRNAs (miRNA) are small noncoding RNAs that neg-atively regulate expression of multiple genes either by induc-ing translational silencing or by causing mRNA degradation(2). It has been firmly established that miRNAs control variouskey cellular processes, such as proliferation, apoptosis, differ-entiation, and development (3), and are implicated in humandiseases, including cancer (4). miRNAs have been identifiedthat function as classic oncogenes or tumor suppressor genes

(4). Genomic deletion or epigenetic silencing of a miRNA thatnormally represses expression of one or more oncogenesmight lead to increased oncogenic expression. Alternatively,amplification, overexpression, or loss of epigenetic silencing ofa gene encoding a miRNA that targets one or more tumorsuppressor genes could inhibit the activity of an antioncogenicpathway (4). Aberrant miRNA profiles have been noted invarious cancers (5, 6), including RCC (7–10). Owing to theirtissue- and disease-specific expression patterns and tremen-dous regulatory potential, miRNAs are being assessed aspotential biomarkers for diagnosis and prognosis of humanmalignancies (11). Recent evidence shows that miRNAs playan important role in the pathophysiology of RCC. Severalstudies have been conducted to identify the RCC-specificmiRNA signature (7–10) though no consensus has beenreached on which miRNAs are relevant for developmentand progression of this malignancy. As the molecular inter-actions of miRNAs with their cognate target genes in varioustumor settings are being explored, the main objective of thisstudy was to identify novel miRNAs that regulate renal car-cinogenesis. In this study, we identified a crucial tumor-suppressive miRNA, miR-708, in renal cancer. Functionalstudies on miR-708 in RCC indicated that miR-708 is aproapoptotic miRNA that regulates renal carcinogenesis.miR-708 is a recently discovered miRNA (12, 13) which hasnot been extensively studied, though a few reports implicatemiR-708 overexpression in lung carcinomas (14, 15) andchildhood acute lymphoblastic leukemias (16). This is thefirst report implicating a tumor suppressor role for thismiRNA in renal cancer, where we show for the first timean important proapoptotic role for miR-708 in renal cancer.

Authors' Affiliation: Department of Urology, Veterans Affairs MedicalCenter, San Francisco and University of California, San Francisco, SanFrancisco, California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Rajvir Dahiya, Urology Research Center (112F),Veteran Affairs Medical Center and University of California, San Francisco,4150 Clement Street, San Francisco, CA 94121. Phone: 415-750-6964;Fax: 415-750-6639; E-mail: rdahiya@urology.ucsf.edu

doi: 10.1158/0008-5472.CAN-11-0073

�2011 American Association for Cancer Research.

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Materials and Methods

Cell lines and cell cultureThe nonmalignant SV-40 immortalized renal cell line HK2

andhuman renal cancer cell lines A498 andCaki2were obtainedfrom the American Type Culture Collection. The HK2 cell linewas maintained in keratinocyte serum-free medium (GIBCOLaboratories) supplemented with 50 mg/mL bovine pituitaryextract, 5% L-glutamine, and 5 ng/mL epidermal growth factor.A498 and Caki2 cells were cultured in Eagle's minimal essentialmedium and McCoy's 5A medium, respectively, each supple-mented with 10% FBS (Atlanta Biologicals) and 1% penicillin/streptomycin (UCSF Cell Culture Facility). All cell lines werecultured in a humidified incubator (5% CO2) at 37�C.

miRNA/siRNA transfectionsCells were plated in growth medium without antibiotics

approximately 24 hours before transfections. Transient trans-fections of miRNA precursor (Ambion)/siRNA (Origene) werecarried out by using Lipofectamine 2000 (Invitrogen) accord-ing to the manufacturer's protocol. miR-708 precursor(PM11161) or negative control (miR-CON; AM17110; Ambion)was used for assays. All miRNA/siRNA transfections were for72 hours.

miRNA microarrayFor miRNA microarray, total RNA was extracted from

HK2, A498, and Caki2 cells by using a miRNeasy mini kit(Qiagen). The miRNA microarray analysis was carried out bya commercial company (Phalanx Biotech), using human v2miRNA OneArray platform that is designed to contain 100%of miRBase Sequence Database Release 15.0.

Tissue samplesTissue samples from radical nephrectomy were obtained

from the San Francisco Veterans Affairs Medical Center(SFVAMC). Informed consent was obtained from all patients.All slides were reviewed by a board-certified pathologist forthe identification of tumor foci as well as normal adjacenttissue. For microdissections, 4-mm slides were hematoxylinand eosin (H&E) stained, reviewed, and marked (tumor andnormal areas) by a board-certified pathologist at the SFVAMC.After that, 10-mm nonstained sections were microdissected byusing aforementioned slides as a template.

RNA and miRNA extractionTotal RNA was extracted from microdissected formalin-

fixed, paraffin-embedded (FFPE) tissues by using a miRNeasyFFPE Kit (Qiagen) and an RNeasy mini kit (Qiagen) was usedfor RNA extraction from cultured cells following the manu-facturer's instructions.

Quantitative real-time PCRMaturemiRNAs and othermRNAswere assayed by using the

TaqMan MicroRNA Assays and Gene Expression Assays, re-spectively, in accordance with the manufacturer's instructions(Applied Biosystems). Samples were normalized to RNU48 orglyceraldehyde-3-phosphate dehydrogenase (GAPDH; Applied

Biosystems), as indicated. The comparative Ct (thresholdcycle) method was used to calculate the relative changes ingene expression on the 7500 Fast Real Time PCR System.

Cell viability, clonability, migratory, and invasionassays

Cell viability was determined at 24, 48, and 72 hours byusing the CellTiter 96 AQueousOne Solution Cell ProliferationAssay Kit (Promega), according to the manufacturer's proto-col. For colony formation assay, cells were counted, seeded atlow density (1,000 cells/plate or 200 cells/plate), and allowedto grow until visible colonies appeared. Then, cells werestained with Giemsa, and colonies were counted. CytoselectCell Migration and Invasion Assay Kit (Cell Biolabs, Inc.) wasused for migration and invasion assays, according to themanufacturer's protocol. Briefly, 48 hours posttransfection,cells were counted and placed on control inserts or Matrigelinserts at 1 � 105 cells/mL in serum-free medium and wereallowed to migrate for 20 hours at 37�C. Cells were removedfrom the top of the inserts and cells that migrated/invadedthough the polycarbonate/basement membrane were fixed,stained, and quantified at optical density (OD) 560 nm afterextraction.

Apoptosis assaysFluorescence-activated cell sorting analysis for apoptosis

was done 72 hours posttransfection, using Annexin V-FITC/7-AAD Kit (Beckman Coulter, Inc.) for apoptosis analysis,according to the manufacturer's protocol. Stained cells wereimmediately analyzed with a flow cytometer (Cell Lab QuantaSC; Beckman Coulter, Inc.).

Adhesion assayAdhesion assays were varied out 72 hours posttransfection

by using a Cytoselect 48-well cell adhesion assay [extracellularmatrix (ECM) array; Cell Biolabs, catalogue no. CBA-070] inaccordance with the manufacturer's protocol. Briefly, cellswere counted and plated at a density of 0.2 � 106 cells/mL,incubated in a tissue culture incubator for 90 minutes to 5hours at 37�C. After incubation, adherent cells were stainedand quantified at OD 560 nm after extraction.

Renal cancer xenograftsWe examined the antitumor effects of miR-708 by local

administration in established tumors as previously described(17, 18). Nude mice (4- to 5-week old; Charles River Labora-tories; n ¼ 12) received subcutaneous injections of 3 � 106

A498 cells in the right flank area in a volume of 100 mL. Oncepalpable tumors developed, caliper measurements were takentwice a week and tumor volume was calculated on the basis ofwidth (x) and length (y): x2y/2, where x < y. When tumorsreached an average volume of 100 to 150 mm3, 6.25 mg ofsynthetic miRNA (miR-708/miR-CON) complexed with 1.6 mLsiPORTamine transfection reagent (Ambion) in 50 mL PBS wasdelivered intratumorally in 3-day intervals. Synthetic miRNAsare double-stranded, ready-to-use miRNA mimics and werepurchased from Ambion, Life Technologies (pre-miR, cata-logue no. AM17100). Mice were killed 2 days after the last

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treatment, tumors were collected, and total RNA wasextracted from tumor tissues for reverse transcriptase PCR(RT-PCR) analysis. All animal care was in accordance with theinstitutional guidelines.

StatisticsAll quantified data represent an average of at least triplicate

samples or as indicated. Data are represented as mean� SEMAll statistical analyses were carried out by StatView (version 5;SAS Institute Inc.). One-way ANOVA or 2-tailed Student's t testwas used for comparisons between groups. The Mann–Whit-ney U test was used to assess the difference between miRNAexpressions in tumor and normal adjacent tissues. c2 test wasused for correlation analysis. Results were considered statis-tically significant at P � 0.05.

Supplementary dataThe supplementary data include supplementary methods,

tables, and figures.

Results

miR-708 expression is attenuated in renal cancerTotal RNA was extracted from normal renal (HK2) and

primary RCC (A498 and Caki2) cell lines and miRNA expres-sion profiling was done to identify dysregulated miRNAs inRCC (data not shown). This screening showed that miR-708 issignificantly downregulated in RCC cell lines. We validated themicroarray data by real-time PCR analysis and confirmed thatmiR-708 was downregulated in primary RCC cell lines(Fig. 1A). To examine the clinical relevance of this finding,we analyzed miR-708 expression in microdissected humanrenal cancer tissues (n ¼ 38) and matched adjacent normalregions by real-time PCR (Fig. 1B). Among these tissues, 28 of38 cases (74%) were clear cell carcinomas (ccRCC), 7 of 38(18%) were of papillary subtype, 3 of 38 (8%) representedgranular cell carcinoma or oncocytoma. While the expressionof miR-708 was unaltered in 8 of 38 cases (21%) and higher in10 of 38 cases (26%), a major fraction of RCC tissue samples(20 of 38, �53%) showed lower miR-708 levels (<75%) relativeto matched normal tissues. The differences were statisticallysignificant with the Mann–Whitney U test (P ¼ 0.0179).Patient and tumor characteristics are summarized in Supple-mentary Table S1. In both the ccRCC and papillary subtypes,approximately 57% of the samples had lower miR-708 expres-sion compared with normal adjacent tissues. We furtherassessed whether miR-708 expression in clinical tissues cor-related with clinicopathologic characteristics such as patho-logic stage and Fuhrman grade (Fig. 1C). Decreased miR-708expression was observed in 55% cases of pT1 and 78% cases ofhigher stage (pT2 þ pT3) RCC. These data suggest that caseswith advanced pathologic stage had decreased miR-708 ex-pression compared with adjacent normal tissues (P ¼ 0.041).However, no statistically significant correlation was observedbetween miR-708 expression and Fuhrman grade. We extend-ed our analysis of clinical specimens by assessing miR-708levels in 20 additional cases of ccRCC and matched normaladjacent tissues by in situ hybridization analysis and again

observed attenuated expression of miR-708 in approximately60% of RCC tissues compared with normal tissues (Fig. 1D).These results suggest a potential tumor suppressor role formiR-708 in renal carcinoma.

Regulation of miR-708 locusHuman miR-708 gene is located at chromosomal position

11q14.1. miR-708 is an intronic miRNA that is located in thefirst intron of a coding gene, ODZ4 (odz, odd Oz/ten-mhomolog 4; Drosophila; Fig. 1E), which is transcribed in thesame direction as miR-708. ODZ4 (also called Teneurin-4) isa homolog of a Drosophila pair-rule gene that encodes atransmembrane protein involved in intercellular signalingduring development (19, 20). To determine whether miR-708is coordinately transcribed with its host gene, we assessedODZ4 expression in the same RCC cell lines by RT-PCR. Wefound that ODZ4 expression is similarly downregulated inA498 and Caki2 cell lines as compared with the nontumori-genic epithelial cell line HK2 indicating that transcription ofmiR-708 may be coupled with its host gene (Fig. 1E). Further,trichostatin A, a pharmacologic inhibitor of histone deace-tylases, increased miR-708 expression alone or in combina-tion with the demethylating agent, 5-aza-20-deoxycytidine(Supplementary Fig. S1), suggesting that this locus is epige-netically regulated primarily by histone modifications inRCC.

miR-708 reexpression suppresses tumorigenicityin vitro

To assess the potential for a tumor suppressive role of miR-708, we reexpressed miR-708 in primary RCC cell lines (A498and Caki2) followed by functional assays. Transient transfec-tion of miR-708 precursor led to overexpression of miR-708as determined by real-time PCR (Fig. 2A). Reexpression ofmiR-708 led to marked morphologic changes in both the celllines (Fig. 2B). Specifically, a pronounced decrease in thefraction of elongated, spindle-shaped cells was paralleled byan increase in rounded, apoptotic cells. The morphologicalterations observed with miR-708 reexpression suggested aprofound increase in apoptotic cells. This was confirmed bystaining with Annexin V-FITC followed by fluorescence mi-croscopy (Supplementary Fig. S2). A significant decrease incell viability was observed over time in A498/Caki2 cellsoverexpressing miR-708 (Fig. 2C) as compared with cellsexpressing control miR (miR-CON). miR-708 reexpression alsodecreased clonogenicity of A498/Caki2 cells compared withmiR-CON (Fig. 2D). Transwell migration and invasion assaysshowed that miR-708 reintroduction decreased the migration(Fig. 2E) and invasion (Fig. 2F) of both cell lines. Theseobservations suggest that miR-708 reexpression suppressesthe tumorigenicity of renal cancer cells in vitro.

Reintroduction of miR-708 induces apoptosis in renalcancer cells

We measured apoptosis in control (mock or miR-CONtransfected) and miR-708–transfected cells by flow cyto-metric analysis of Annexin V-FITC/7-AAD–stained A498and Caki2 cells (Fig. 3A and B). It was observed in both

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Figure 1. miR-708 expression is attenuated in renal cancer. A, quantitative RT-PCR analysis of relative miR-708 expression levels in human renal cancer(A498 and Caki2) and normal immortalized renal cell line (HK2). Data were normalized to RNU48 control and are represented as mean � SEM. B,relative miR-708 expression levels in RCC clinical specimens and patient-matched normal tissues as assessed by real-time PCR. The table summarizesthe relative miR-708 expression levels in these specimens. C, correlation of miR-708 expression with subtype, clinicopathologic stage, and Fuhrman grade ofrenal cancer tissues used for miR-708 expression analysis in (B). D, in situ hybridization analysis of miR-708 expression in human renal cancer tissuesandmatched cancer adjacent normal tissues showing attenuated miR-708 expression in RCC tumor (bottom) compared with matched normal adjacent tissue(top). U6 staining confirmed the preservation of intact small RNAs in the same tissues (middle); H&E stained sections allowed the identification of tumors (right).E, schematic representation of the genomic location of the miR-708 locus in humans at chromosomal position 11q14.1 in ODZ4 gene intron. Relativeexpression levels of ODZ4 in RCC cell lines as assessed by real-time PCR. *, P < 0.05.

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the cell lines that the average apoptotic cell fractions (earlyapoptotic þ apoptotic) were significantly increased uponmiR-708 reexpression compared with miR-CON (P < 0.001)or mock-transfected cells (P < 0.001) with a concomitantdecrease in the viable cell population. This points to aproapoptotic role of miR-708 and suggests that miR-708affects apoptotic pathways in regulating tumorigenicity.

We also examined the expression of various apoptoticcomponents by Western blot analysis and found thatmiR-708 reexpression leads to induction of TNF-relatedapoptosis-inducing ligand (TRAIL; Fig. 3C). Also, cleavedcaspase-3 and caspase-7 and cleaved PARP were detected byimmunoblot analysis (Fig. 3C), further supporting the factthat miR-708 is a proapoptotic miRNA.

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Figure 2.Restoration of miR-708 expression suppresses tumorigenicity in vitro. A, relative miR-708 expression in A498/Caki2 cells transfected withmiR-CON/miR-708/mock–transfected cells as assessed by real-time PCR. B, morphologic alterations in A498/Caki2 cells uponmiR-708 restoration assessed by phase-contrast microscopy. Cellular viability assay (C), colony formation assay (D), transwell-migration assay (E), and invasion assay (F) in A498/Caki2 cellsmock transfected or transfected with miR-CON or miR-708. *, P < 0.05.

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Figure 3. miR-708 induces apoptosis and reduces adherence to ECM. A, apoptosis assay in A498/Caki2 cells after miR-CON (left) and miR-708(right) treatments. B, representation of average apoptotic fractions (early þ late apoptotic) in each group. *, P < 0.0001. C, immunoblot analysis forapoptotic markers in mock/miR-CON/miR-708–transfected A498 cells. GAPDH was used as a loading control. D, adhesion assay for miR-CON/miR-708–transfected A498 and Caki2 cells to following ECM components: fibronectin (FN), collagen I (col I), collagen IV (col IV), laminin (LN), fibrinogen (FB).BSA was used as negative control. *, P < 0.05.

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miR-708 expression reduces adherence of renal cancercells to extracellular matrix

In view of the observed effects of miR-708 reexpression onthe morphology of renal cancer cells and its effect on tumor-igenicity, we monitored the ability of miR-708–overexpressingcells to attach to the ECM with in vitro adhesion assays. It wasobserved that miR-708 reintroduction led to decreased abilityto attach to a range of ECM components as compared withcontrol cells (Fig. 3D). However, adhesion to the negativecontrol bovine serum albumin (BSA) did not differ betweenmiR-708–overexpressing cells and control cells (Fig. 3D).

Intratumoral delivery of miR-708 leads to regression oftumors in a renal cancer xenograft model

Because the in vitro data showed an antitumorigenic role formiR-708 in RCC, we examined the therapeutic potential ofsynthetic miR-708 mimics in vivo in a mouse renal cancerxenograft model. Nude mice were subcutaneously inoculatedwith A498 cells and maintained until the tumor cells hadformed solid, palpable tumors with an average volume of100 to 150 mm3. Thirty days following inoculation, miR-708or a negative control miRNA was repeatedly administered by

intratumoral injections every 3 days. Allmicewere killed onday58. As shown in Fig. 4A andB, intratumoral delivery of syntheticmiR-708 induced a specific inhibitory response and robustlyinterfered with tumor growth compared with control mice. Tocorrelate the therapeutic response with delivery of miR-708,RNA was extracted from harvested tumors and miR-708 ex-pression was assessed by quantitative RT-PCR. Tumorsinjected with miR-708 mimic contained approximately5,000-fold more miR-708 than control tumors (Fig. 4C).

miR-708 targets a cohort of genes in renal cancer cellsTo identify effectors of miR-708, we used the miRANDA (21)

algorithm that predicts the mRNA targets of a miRNA. Guidedby the target prediction algorithms, we carried out Westernblot analysis for putative miR-708 targets in A498 cells thatwere either mock transfected or transfected with miR-708/miR-CON (Fig. 5A). Consistent with the observed effects ofmiR-708 on apoptosis and cellular adhesion, we foundkey molecules of these cellular processes as direct targetsof miR-708. miR-708 overexpression led to decreased proteinlevels of survivin, a small inhibitor of apoptosis protein that isdifferentially expressed in cancer (22) and plays a pivotal role

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Figure 4. Intratumoral delivery ofmiR-708 leads to regression oftumors in a renal cancer xenograftmouse model. A, A498 cells weresubcutaneously injected into nudemice to form solid, palapabletumors (day 30), following whichsynthetic miR-708 or miR-CONwere intratumorally delivered for 4weeks. Tumor volumes followingmiR-708 administration weresignificantly reduced. *, P < 0.05.B, representative images of micefrom the 2 groups beforeintratumoral injections werestarted (day 30) and on day 58 areshown. Subcutaneous tumors areindicated by arrows. C, relativemiR-708 expression in renalcancer xenografts as assessed byreal-time PCR. The average valuesare represented. *, P < 0.05.

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in renal cancer progression and metastasis (23, 24). Also, miR-708 repressed the protein levels of melanoma cell adhesionmolecule (MCAM) which plays an important role in malignantprogression and tumor metastasis (25).Interestingly, miR-708 repressed the protein levels of tran-

scription factorZEB2, a transcriptional repressor that regulatesthe expression of E-cadherin and epithelial-to-mesenchymaltransition (EMT; 26, 27). In keeping with this novel finding,examination of E-cadherin levels in miR-708–transfected cellsshowed that miR-708 reexpression augments the levels of this

epithelial marker and represses fibronectin. Also, miR-708inhibited the expression of polycomb repressor, BMI1. Weinvestigated whether the 30-UTR of ZEB2 and BMI1 are func-tional targets ofmiR-708 in renal cancer. Transient transfectionof human A498 cancer cells with the respective 30-UTR plas-mids along with different concentrations of miR-708 precursorled to a significant decrease in promoter activity when com-pared with the control vector (Fig. 5B and C) suggesting thatmiR-708 directly represses these genes. The luciferase activityof the reporter vectors containing a mutated 30-UTR of the

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3′ GGGUCGAU----CUAACAUUCGAGGAA 5′

263: 5′… AGAAGCUAUUUAUUAUGAGCUUCUA… 3′ : :

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3′ GGGUCGAUC-----UAACAUUCGAGGAA 5′

5: 5′… GGCAGCGGGUCCUGGGAUGCUCCUG…3′ :

BMI1

ZEB2

FN1

MCAM

GAPDH

E-cadherin

Normalized intensities

Mock miR-CON miR-708

Survivin 0.93 0.93 0.50

MCAM 1.15 1.14 0.78

FN1 1.60 1.36 0.87

ZEB2 1.05 0.98 0.74

E-cadherin 0.19 0.17 0.44

BMI1 0.75 0.60 0.37

% change

46%

32%

25%

38%

−5%

miR-CON miR-708 miR-CON miR-708

25 nmol/L 50 nmol/L 100 nmol/L

miR-CON miR-708 miR-CON miR-708 miR-CON miR-708

25 nmol/L 50 nmol/L 100 nmol/L

259%

36%

* *

* **

Figure 5. miR-708 targets a cohort of genes in renal cancer cells. A, immunoblots for endogenous survivin, MCAM, FN1, ZEB2, E-cadherin, and BMI1protein in A498 cells transfected as indicated. GAPDH was used a loading control. Quantifications of immunoblots are represented in the table andpercent changes in miR-708 transfectants relative to miR-CON are also indicated. Schematic representation of ZEB2 30-UTR (B) and BMI1 30-UTR (C)showing putative miR-708 target sites and luciferase activity assay with respective wild-type and mutant luciferase constructs and control constructcotransfected with increasing concentrations of miR-CON/miR-708. For all luciferase activity assays, firefly luciferase values were normalized to renillaluciferase activity and plotted as relative luciferase activity. *, P < 0.05.

miR-708 in Renal Cancer

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respective genes was unaffected by miR-708. These observa-tions led us to conclude that miR-708 regulates a cohort ofgenes which play an important role in cellular survival, adhe-sion, invasion, and metastasis.

Survivin is a direct target of miR-708miR-708 significantly reduced the expression (Fig. 5A) of

survivin in A498 cells. The 30-UTR of survivin mRNA has 3putative miR-708 binding sites (Fig. 6A). We carried outluciferase reporter assays with the survivin construct inmiR-203/miR-CON–expressing A498 cells (Fig. 6A) and ob-served a consistent reduction of luciferase activity upon miR-708 transfection suggesting that miR-708 represses survivindirectly. We also examined expression of survivin mRNA in 12

pairs of human tissue samples from Fig. 1B to determinewhether there was an inverse relationship between survivinand miR-708 expression. The relative level of survivin expres-sion in normal versus tumor tissue is shown in Fig. 6B.Survivin expression was inversely correlated with miR-708expression in these tested samples (P ¼ 0.0001), furthersupporting the idea that miR-708 directly regulates survivinlevels in RCC. To further validate survivin as a direct target, weextracted tumor RNA from the renal cancer xenograft mousemodel study (Fig. 4), and asked whether intratumoral admin-istration of miR-708 alters survivin expression. Analysis ofrelative survivin expression in these tumors showed that miR-708–injected tumors show a lower average expression ofsurvivin than control tumors (Fig. 6C).

0

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hsa-miR-708

Survivin 3′-UTRSite 1

Site 2

Site 3

3′ GGGUCGAUCU—AACAUUCGAGGAA 5′

556: 5′… UGGAGACAGAGUCCCUGGCUCCUC… 3′:

794: 5′… GGGGCACAUGCUGGCCGCUCCUC… 3′

3′ GGGUCGAUCUAACAUUCGAGGAA 5′

3′ GGGUCGAUC-UA—ACAUUCGAGGAA 5′

1,304: 5′… ACCUGUUUGUAUCAUCCGGGCUCCUU… 3′

hsa-miR-708

::hsa-miR-708

0.2

0

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1.0

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2 3 7 8 14 16 21 23 26 28 37 38Patient no.

Survivin 3′-UTR

Survivin 3′-UTR

*

*

Figure 6. Survivin is a direct target ofmiR-708. A, schematic representation ofsurvivin 30-UTR showing the relativepositions of 3 putative miR-708 targetsites. Survivin 30-UTR constructencompassing these sites or the controlconstruct was cotransfected into A498cells along with miR-708/miR-CON andassayed for relative luciferase activity.*, P < 0.05. B, relative survivin expressionin human RCC tumor tissues and patient-matched normal tissues as assessed byreal-time PCR. For this analysis, 12 of 38pairs of human tissue samples used inFig. 1B of this study (indicated by patientno.) were used to determine whether therewas an inverse relationship betweensurvivin and miR-708 expression. C,relative survivin expression in harvestedtumors from mouse renal cancerxenografts intratumorally injected withmiR-CON/miR-708. Survivin expressionfrom the 2 groups was averaged andplotted. *, P < 0.05.

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Survivin knockdown partially phenocopies miR-708reexpression in renal cancer cellsIn view of the observed profound effects of miR-708 on

apoptosis, we sought to determine whether miR-708 mediatesits antitumorigenic effects primarily through survivin. Toaddress this, we treated A498 cells with survivin siRNA fol-lowed by functional assays. RT-PCR and immunoblot analysisconfirmed specific knockdown of survivin by siRNA (Fig. 7A).As expected, survivin siRNA-transfected cells assumed arounded, apoptotic morphology (Fig. 7B) similar to the phe-notype observed upon miR-708 overexpression in A498 cells.Apoptosis assay showed that apoptotic cell fractions (earlyapoptotic þ apoptotic) were significantly increased uponsurvivin knockdown compared with control siRNA-treatedcells (P ¼ 0.018) as observed upon miR-708 reexpression(Fig. 7C). Treatment with survivin siRNA also led to decreasedproliferation and clonogenic survival of A498 cells (Supple-mentary Fig. S3). These results suggest that the growth-suppressive effects of miR-708 are in part facilitated bysurvivin downregulation.

Discussion

In this study, we identify a crucial tumor-suppressivemiRNA, miR-708, that plays an important role in renal carci-nogenesis. Several prior studies have reported on the dysre-gulation of various miRNAs in RCC (7–10). However, this is thefirst report that implicates miR-708 in the pathogenesis of

RCC. In contrast to nontumorigenic immortalized renal ep-ithelial cells, tumorigenic cell lines A498 and Caki2 containedlow amounts of miR-708, suggesting that miR-708 may havetumor-suppressive activity. Assessment of miR-708 in humankidney tissues also pointed to a statistically significant atten-uation of miR-708 expression in approximately 50% to 60% ofRCC cases. A limitation to our study was the relatively smallnumber of clinical samples at our disposal. Further studieswith more clinical samples are warranted.

In addition, our in vitro and in vivo data suggest thatreexpression of miR-708 suppresses tumorigenicity confirm-ing the tumor-suppressive role of miR-708 in RCC.

Apoptosis is a well-orchestrated cellular mechanism thatbalances cell proliferation and cell death. In fact, the ability toevade apoptosis is a hallmark of tumorigenesis. Here wediscovered an important proapoptotic role of miR-708.miR-708 reexpression led to dramatic induction of apoptosisconcomitant with cleavage of caspase-3 and caspase-7 in RCCcell lines. Also, our study suggests that miR-708 is a regulatorof death receptor TRAIL that selectively induces apoptosis intumor-derived cell types through caspase activation and is apromising antitumor agent (28). Recent findings show thatmiRNAs regulate death receptors and pro- and antiapoptoticgenes involved in programmed cell death pathways (29, 30). Ina large scale screening for gene networks regulating TRAIL-induced apoptosis, Ovcharenko and colleagues transfectedapproximately 200 synthetic miRNAs in MDA-MB-453 breastcancer cells and identified that miR-28, miR-10a, 196a, and

Figure 7. Survivin knockdownpartially phenocopies miR-708reexpression in renal cancer cells.A498 cells were transfected withsiRNA specific to survivin and acontrol nonspecific (NS) siRNA for72 hours followed by variousassays. A, relative survivin mRNAexpression (top) and proteinexpression (bottom) after siRNAtransfections as assessed by real-time PCR and immunoblotting,respectively. B, morphologicalterations observed after survivinknockdown in A498 cells asassessed by phase-contrastmicroscopy. C, apoptosis assay inA498 cells after NS siRNA (left) orsurvivin siRNA (right) treatments.

A

NS siRNA Survivin siRNA

NS siRNA Survivin siRNA

C

B

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siR

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rviv

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iRN

A

Survivin

GAPDH

% Apoptotic 3 ± 1% Early apoptotic 2 ± 1% Viables 94 ± 2% Dead 1 ± 2

% Apoptotic 11 ± 2% Early apoptotic 12 ± 1% Viables 76 ± 2% Dead 1 ± 1

Total cells Total cells

miR-708 in Renal Cancer

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miR-337 led to induction of caspase-3 activity (31). In thisstudy, we found that miR-708 is a novel miRNA that regulatesapoptosis in renal cancer cells. In addition, miR-708 impairsthe ability of renal cancer cells to attach to ECM components.

Our results show that miR-708 can pleiotropically regulate acohort of genes that play a role in cellular survival, adhesion,invasion, and metastasis. Among the putative targets of miR-708, we have validated that survivin is an important target.Survivin knockdown studies suggest that the proapoptoticrole of miR-708 may be mediated primarily through regulationof survivin. In addition, lower expression of survivin in miR-708 xenografts provide evidence that the mechanism weidentified in cell lines is repeatable in xenograft tumor cells.miR-708–mediated regulation of survivin is a highly significantfinding as this nodal protein orchestrates extensive, tumor-specific signaling networks and is an attractive drug target(32). Survivin is a regulator of cellular homeostasis, modulat-ing cell death, survival, the cell cycle, and microtubule dy-namics (22). Survivin expression is an independent predictorof ccRCC progression and death from RCC (33). Survivinexpression has been correlated with several adverse patho-logic features in ccRCC including tumor size, nuclear grade,tumor-node-metastasis (TNM) classification, presence of met-astatic disease, coagulative tumor necrosis, and sarcomatoiddifferentiation. Thus, survivin has the potential to be impor-tant in disease prognosis and to serve as a novel target for thedevelopment of new adjuvant therapies (34).

Interestingly, we found that BMI1 and ZEB2 are targets ofmiR-708. BMI1, a member of the polycomb-repressive com-plex 1 (35, 36) is frequently overexpressed in cancers (37–39).Overexpression of BMI1 in carcinoma cell lines resulted inthe acquisition of EMT characteristics, induction of stem-cell markers and enhancement of tumor-initiating capability(40). ZEB2, a repressor of E-cadherin (CDH1), coordinatesEMT, a key embryonic process (41) that is reactivated duringtumorigenesis. Recent evidence indicates that ZEB tran-scription factors may also regulate apoptosis and senes-cence apart from regulation of EMT (42, 43). Our resultssuggest that miR-708 reexpression induces apoptosis andalso causes decreased motility and invasiveness. Because ofthe predominant and dramatic proapoptotic effects of miR-

708, decreased migratory and invasive properties may becaused partly by effects on apoptosis. However, our presentresults showing that in addition to targeting survivin, miR-708 also causes reduced expression of EMT regulators ZEB2and BMI1 concomitant with induction of E-cadherin expres-sion and repression of fibronectin expression suggests thatmiR-708 may also affect migration and invasion directly. Arecent study suggests that miR-200c represses ZEB1 andZEB2 and also regulates induction of apoptosis through thedeath receptor CD95 (44).

In conclusion, our study identifies miR-708 as an importantmiRNA that regulates renal carcinogenesis through a cohort ofeffectors. This study shows that miR-708 plays an importantrole as a proapoptotic miRNA in renal cancers. In view of ourpresent results showing an attenuation of miR-708 expressionin human RCC clinical specimens and the suppression oftumorigenicity upon restoration of miR-708 expression, wehypothesize that miR-708 may be an attractive target forprognostic and therapeutic interventions in RCC. Further-more, our in vivo data showing growth inhibition of estab-lished renal tumor xenografts by intratumoral delivery ofsynthetic miR-708 oligonucleotides support the therapeuticpotential of this novel miRNA in RCC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Roger Erickson for his assistance with the preparation of themanuscript.

Grant Support

This study was supported by Grants T32DK007790, RO1CA138642, andRO1CA130860 (NIH), VA Research Enhancement Award Program, and MeritReview grants.

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

Received January 10, 2011; revised June 27, 2011; accepted July 23, 2011;published OnlineFirst August 18, 2011.

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2011;71:6208-6219. Published OnlineFirst August 18, 2011.Cancer Res   Sharanjot Saini, Soichiro Yamamura, Shahana Majid, et al.   Tumorigenicity in Renal Cancer CellsMicroRNA-708 Induces Apoptosis and Suppresses

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