Induction of miR-137 by Isorhapontigenin (ISO) Directly ... · Sp1/Cyclin D1 expression, induction...

Cancer Biology and Signal Transduction

Induction of miR-137 by Isorhapontigenin (ISO)Directly Targets Sp1 Protein Translation andMediates Its Anticancer Activity Both In Vitroand In VivoXingruo Zeng1,2, Zhou Xu1, Jiayan Gu3, Haishan Huang1,3, Guangxun Gao1, Xiaoru Zhang1,Jingxia Li1, Honglei Jin1, Guosong Jiang1, Hong Sun1, and Chuanshu Huang1

Abstract

Our recent studies found that isorhapontigenin (ISO)showed a significant inhibitory effect on human bladder cancercell growth, accompanied with cell-cycle G0–G1 arrest as well asdownregulation of Cyclin D1 expression at transcriptional levelvia inhibition of Sp1 transactivation in bladder cancer cells. Inthe current study, the potential ISO inhibition of bladder tumorformation has been explored in a xenograft nude mouse model,and the molecular mechanisms underlying ISO inhibition ofSp1 expression and anticancer activities have been elucidatedboth in vitro and in vivo. Moreover, the studies demonstratedthat ISO treatment induced the expression of miR-137, whichin turn suppressed Sp1 protein translation by directly targetingSp1 mRNA 30-untranslated region (UTR). Similar to ISO treat-ment, ectopic expression of miR-137 alone led to G0–G1 cell

growth arrest and inhibition of anchorage-independent growthin human bladder cancer cells, which could be completelyreversed by overexpression of GFP-Sp1. The inhibition ofmiR-137 expression attenuated ISO-induced inhibition ofSp1/Cyclin D1 expression, induction of G0–G1 cell growtharrest, and suppression of cell anchorage-independent growth.Taken together, our studies have demonstrated that miR-137induction by ISO targets Sp1 mRNA 30-UTR and inhibits Sp1protein translation, which consequently results in reduction ofCyclin D1 expression, induction of G0–G1 growth arrest, andinhibition of anchorage-independent growth in vitro and in vivo.Our results have provided novel insights into understandingthe anticancer activity of ISO in the therapy of human bladdercancer. Mol Cancer Ther; 15(3); 512–22. �2016 AACR.

IntroductionBladder carcinoma is highly prevalent and the second most

common genitourinary malignant disease in the United States(1). Bladder carcinoma is threatening for human beings when itinvades muscles. Among all the cancer types, bladder carcinomaranks the fifth in total healthcare cost with an annual expenditureof approximately $4 billion in the United States alone (2).Although MVAC (methotrexate, vinblastine, adriamycin, andcisplatin) chemotherapy has been widely used for treatment ofadvanced bladder cancers, it is accompanied withmajor toxic sideeffects (3). Therefore, development of less toxic alternate chemo-therapeutic therapies and/or dietary management strategies isof high significance for prevention and therapy of this disease(2, 3). Isorhapontigenin (ISO) is a new derivative of stilbenecompound isolated from Chinese herb Gnetum cleistostachyum,

and its chemical structure is shown in our previous publications(4, 5). Recently, our group has reported that ISO effectivelysuppresses bladder cancer cell growth in vitro (4, 5) and has foundthat ISO treatment induces G0–G1 cell growth arrest and inhibitsanchorage-independent growth of human bladder cancer cellsthrough downregulated Cyclin D1 gene transcription via inhibi-tion of Sp1 transactivation in bladder cancer cells (4).

Sp1 is the first transcription factor to be isolated from mam-malian cells and belongs to the specificity Protein/Kruppel-likeFactor (SP/KLF) family (6), which are characterized by theirCOOH-terminal domains containing threeC2H2-type zincfingersthat recognize GC-richmotif in the promoters of their target genes(7). Sp1 is ubiquitously expressed in various mammalian cellsand plays an important role in the regulation of numerous genesinvolved in various cellular processes (8), such as cell differenti-ation, cell growth, and apoptosis. An increasing number ofevidence shows that Sp1 is upregulated in many cancer tissues,including breast carcinomas (9), hepatocellular carcinomas (10),thyroid cancer (11), colorectal cancer (12), pancreatic cancer (13),gastric cancer (14), and lung cancer (15). Furthermore, Sp1expression is also increased in the bladder epithelium of themouse exposed to n-butyl-N-(4-hydroxybutyl)-nitrosamine, awell-characterized mouse carcinogen for invasive bladder cancerinduction (16). Sp1 expression increases by 8- to 18-fold inmalignant transformed fibroblasts, whereas knockdown of Sp1expression blocks the tumorigenicity of transformedfibroblasts inxenografts athymic nude mouse model (17). It has been reportedthat the upregulation of Sp1 is also associated with poor clinical

1Nelson Institute of Environmental Medicine, New York University,School of Medicine, Tuxedo, New York. 2Department of Nephrology,Central Hospital of Wuhan, Wuhan, China. 3Zhejiang Provincial KeyLaboratory for Technology and Application of Model Organisms,School of Life Sciences, Wenzhou Medical University, Wenzhou,Zhejiang, China.

Corresponding Author: Chuanshu Huang, New York University School ofMedicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: 845-731-3519; Fax:845-351-2320; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0606

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

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prognosis among patients with gastric and pancreatic cancer(14, 18, 19), suggesting that Sp1 may act as an oncoprotein intumor development. The pro-oncogenic activity of Sp1 is primar-ily due to Sp1-regulated genes, which include several genes thatplay pivotal roles in cancer cell proliferation (Cyclin D1, EGFR),survival (survivin, bcl-2), angiogenesis [VEGF and its receptors(VEGFR1 and VEGFR2)], and inflammation (NF-kB, p65;refs. 4, 20). Thus, Sp1 is considered as an important target formechanism-based anticancer drugs. Our previous studies haverevealed that ISO acts as a novel mechanism-based cancer ther-apeutic agent against human bladder cancer by inhibition of Sp1transactivation in different human bladder cancer cell lines (4, 5).However, the anticancer effect of ISO in vivo and the molecularmechanisms underlying ISO inhibition of Sp1 expression hasnever been explored to the best of our knowledge. In currentstudies, we explored the ISO inhibition of human bladder tumorformation in xenografts athymic nude mouse model and themolecular mechanisms underlying ISO suppression of Sp1expression both in vitro and in vivo.

Materials and MethodsPlasmids, antibodies, and reagents

The Sp1 expression construct, pEGFP-Sp1, andmiR-137 expres-sion construct, pcDNA3.2/V5-mmu-mir-137, were obtained fromAddgene. Human Sp1 30-untranslated region (UTR) luciferasereporter, being cloned into the pGL3-control luciferase assayvector, was kindly provided by Dr. Guido Marcucci from Depart-ment of Medicine, Ohio State University, Columbus, OH (21).Sp1 30-UTR point mutation was amplified from wild-type (WT)template by overlap PCRusing primers: forward: 50-GATCTTTGC-TAGGACATCCTAAATTTATATACTT-30; reverse: 50-AAGTATA-TAAATTTAGGATGTCCTAGCAAAGATC-30. The miR-145 expres-sion construct, pBluescript-miR-145 (hsa-miR-145), was kindlyprovided by Dr. Renato Baserga from Department of CancerBiology, Thomas Jefferson University, Philadelphia, PA (22). ThemiR-137 inhibitor expression plasmid (HmiR-AN0175-AM03)was purchased from Genecopoeia. The antibody against b-actinwas bought from Cell Signaling Technology. The antibodiesagainst Cyclin D1, Sp1, Sp4, and GAPDH were bought fromSanta Cruz Biotechnology. ISO with purity more than 99% waspurchased from Roche Pharma and was dissolved in DMSO tomake a stock concentration at 20 mmol/L.

Cell culture and transfectionHuman bladder cancer cell line UMUC3 was provided by

Dr. Xue-Ru Wu (Departments of Urology and Pathology, NewYork University School of Medicine, New York, NY) in 2010 asdescribed in our previous studies (5). T24T was kindly providedby Dr. Dan Theodorescu (Department of Urology, University ofVirginia, Charlottesville, VA) in 2010 and used in our previousstudies (4, 5, 23). The UMUC3 cells were cultured in DMEMsupplemented with 10% FBS, 2 mmol/L L-glutamine, and25 mg/mL of gentamycin, and the T24T cell was cultured in a1:1 mixture of DMEM/Ham's F12 medium supplemented with5% FBS, 2 mmol/L L-glutamine, and 25 mg/mL of gentamycin.All cell lines were subjected to DNA tests and authenticated inour previous studies (4). Both cell lines are regularly authen-ticated on the basis of viability, recovery, growth, morphology,and chemical response as well, and were most recently con-firmed 4 to 6 months before use by using a short tandem repeat

method. The transfections were carried out using PolyJet DNAIn Vitro Transfection Reagent (SignaGen Laboratories) accord-ing to the manufacturer's instructions. The stable transfectionselection of Sp1, miR-137, and miR-145 in UMUC3 and T24Tcells was subjected to neomycin selection for 4 to 6 weeks,whereas miR-137–specific inhibitor stable transfectants wereselected by hygromycin for 4 to 6 weeks. The survived stabletransfectants were pooled as stable mass culture as described inour previous studies (24, 25).

Western blottingCells were extracted with cell lysis buffer (10 mmol/L Tris-HCl,

pH 7.4, 1% SDS, and 1 mmol/L Na3VO4), and protein concen-trations were determined by NanoDrop 2000 spectrophotometer(ThermoScientific). The cell extracts were subjected to SDS-PAGE,transferred to polyvinylidene fluoride membranes (Bio-Rad),probed with the indicated primary antibodies, and incubatedwith the alkaline phosphatase (AP)-conjugated secondary anti-body. The protein band specifically bound to the primary anti-body was detected by Typhoon FLA 7000 (GE Healthcare) usingan alkaline phosphatase–linked secondary antibody and anenhanced chemifluorescence Western blotting system asdescribed in our previous studies (5, 23).

Anchorage-independent growth assayAnchorage-independent growth in soft agar (soft-agar assay)

was performed as described in our earlier studies. Briefly, the1 � 104 cells mixed with ISO at final concentration of 10 mmol/L or vehicle control in 10% FBS Basal Medium Eagle (BME)containing 0.33% soft agar and were seeded over the basal layercontaining 0.5% agar containing 10% FBS/BME in each well of6-well plates. The plates were incubated in 5%CO2 incubator at37�C for 3 weeks. Colonies were captured under a microscope,and only colonies with over 32 cells were counted. The resultswere presented as mean� SD obtained from three independentexperiments.

Cell-cycle analysisThe cells were treated with ISO at 10 mmol/L or control vehicle,

and the cells were then harvested and fixed in 75% ethanolovernight. The cellswere suspended in the staining buffer contain-ing 0.1% Triton X-100, 0.2 mg/mL RNase A, and 50 mg/mLpropidium iodide at 4�C. DNA content was then determined bya flow cytometry Epics XL flow cytometer (BeckmanCoulter Inc.),and the results were analyzed with EXPO32 software.

RT-PCRCells were treated with 10 mmol/L of ISO and were then

extracted for total RNA using TRIzol reagent (Invitrogen), accord-ing to the manufacturer's instructions. The cDNAs were synthe-sized with the Thermo-Script RT-PCR system (Invitrogen). ThemRNA was evaluated by semiquantitative RT-PCR. The primersfor human sp1 were 50-ATTAACCTCAGTGCATTGGGTA-30 and50-AGGGCAGGCAAATTTCTTCTC-30. The primers for humanb-actin were 50-AGAAGGCTGGGGCTCATTTG-30 and 50-AGGGGCCATCCACAGTCTTC-30. The PCR products were sepa-rated on 2%agarose gels and stainedwith ethidiumbromide, andthe results were imagined with Alpha Innotech SP Image system(Alpha Innotech Corporation).

miR-137 Regulation of Sp1 Protein Translation

www.aacrjournals.org Mol Cancer Ther; 15(3) March 2016 513




Quantitative RT-PCR for miRNA assayTotal miRNAs were extracted using the miRNeasy Mini Kit

(Qiagen). Total RNA (1 mg) was used for reverse transcription,and the miRNA expression was determined by the 7900HT FastReal-time PCR system (Applied Biosystems) using the miScriptPCR Kit (Qiagen). The primer for miRNA was purchased fromInvitrogen, and U6 was used as a control. Cycle threshold (CT)valueswasdetermined, and the relative expressionofmiRNAswascalculated by using the values of 2�DDCT.

(35S) methionine pulse assaysT24T cells were cultured in each well of 6-well plate till

70% to 80% confluence, and the cell culture medium wasreplaced with 0.1% FBS DMEM and incubated for another24 hours. The cells were then treated with 10 mmol/L ofISO diluted in 2% FBS methionine/cysteine-free DMEM con-taining 35S-labeled methionine/cysteine (250m Ci per dish,Trans 35S-label, ICN) for the indicated time periods. The cellswere extracted with lysis buffer (Cell Signaling Technology)containing complete proteinase inhibitor mixture (Roche).Total lysate of 500 mg was incubated with anti-Sp1 anti-

body-conjugated agarose beads (R&D Systems) at 4�C over-night. The immunoprecipitate was washed with the cell lysisbuffer five times and heated at 100�C for 5 minutes after finalwashing. The protein samples were then subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis analysis.35S-labeled Sp1 protein was imaging captured with the Phos-phorImager (Molecular Dynamics).

Cancer tissue specimensTwenty-six pairs of primary invasive bladder cancer specimens

and their paired adjacent nontumorous bladder tissues wereobtained from patients who underwent radical cystectomy atDepartment of Urology of the Union Hospital of Tongji MedicalCollege between 2012 and2013. All specimenswere immediatelysnap-frozen in liquid nitrogen after surgical removal. Histologicand pathologic diagnoses were confirmed by a pathologist basedon the 2004WorldHealthOrganizationConsensus Classificationand Staging System for bladder neoplasms. All specimens wereobtained with appropriate informed consent from the patients,and the approval was obtained from the Medical Ethics Com-mittee of Tongji Medical College, China.

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Figure 1.ISO treatment inhibited human bladder tumor growth accompaniedwith reduction of Sp1 and Cyclin D1 protein expression in xenograft nudemice. A and B, athymicnude mice were subcutaneously injected with T24T cells in the right axillary region and received intraperitoneal injection with ISO at dose of 150 mg/kg bodyweight or vehicle control as indicated in the Materials and Methods section. Six weeks after ISO treatment, the mice were sacrificed, and the tumor wassurgically removed and photographed (A) as well as weighed (B). C–E, the representative IHC images showing expression of Sp1 and Cyclin D1 in bladdercancer tissues collected from nude mice. F, the Sp1 protein expression was positively correlated with tumor weight in nude mice. G, the representative IHCimages exhibiting the positive correlation between Sp1 and Cyclin D1 expression in bladder cancer tissues from nude mice.

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Mol Cancer Ther; 15(3) March 2016 Molecular Cancer Therapeutics514




Luciferase assayFor the determination of Sp1 30-UTR luciferase reporter activity,

the cells were transiently cotransfected with Sp1 30-UTR luciferasereporter and TK. The transient transfectants were seeded into eachwell of 96-well plates (1 � 104 cells per well) and cultured for24 hours. The cells were treated with ISO (10 mmol/L) for theindicated times and then extracted with lysis buffer (25 mmol/LTris-phosphate, pH 7.8, 2 mmol/L EDTA, 1% Triton X-100, and10% glycerol), and the luciferase activity was determined by themicroplate luminometer (Microplate Luminometer LB 96V; Bert-holdGmbH&Co.) using the luciferase assayKit (PromegaCorp.).

Tumor xenografts and in vivo ISO treatmentAll animal studies were performed in the animal institute of

WenzhouMedicalUniversity according to the protocols approvedby the Medical Experimental Animal Care Commission of Wenz-hou Medical University. The 12 female athymic nude mice (3–4weeks old) were purchased from Shanghai Silaike ExperimentalAnimal Company, Ltd. (license No. SCXK, Shanghai 2010—0002), and themice at age of 5 to 6 weeks were randomly dividedinto two groups and were then subcutaneously injected with0.2 mL of T24T cells (2 � 106 suspended in 100 mL PBS) in theaxillary region. The mice of ISO group received i.p. injection of150 mg/kg ISO every other day, starting at day one after cellinoculation, whereas control mouse received vehicle only. Thenude mice were maintained under sterile conditions according to

the protocol of the American Association for the Accreditation ofLaboratory Animal Care. These mice were evaluated twice a weekfor the appearance and size of tumors, and tumors weremeasuredwith calipers to estimate the volume. Tumor sizes were evaluatedusing the formula: Volume (mm3) ¼ [width2 (mm2) � length(mm)]/2. Six weeks after ISO treatment, the mice were sacrificedand the tumors were surgically removed, photographed, weighed,and used for further pathologic and histopathologic evaluation.No mouse died or was sacrificed before the end of the in vivoexperiment.

ImmunohistochemistryTumor tissues obtained from the sacrificedmice were formalin-

fixed and paraffin-embedded. For IHC staining, we used anti-bodies specific against Sp1 (1:30; Santa Cruz Biotechnology) orCyclin D1 (1:200; Santa Cruz Biotechnology). The resultantimmunostaining images were captured using the Axio VisionRel.4.6 computerized image analysis system (Carl Zeiss). Proteinexpression levels were analyzed by calculating the integratedoptical density per stained area using Image-Pro Plus version6.0 (Media Cybernetics). More detailed procedure was describedin our previous published studies (26).

Methylation-specific PCRGenomicDNAwas isolatedwith theDNeasy Blood&TissueKit

(Qiagen) according to the manufacturer's instruction. Genomic

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Figure 2.Sp1 downregulation mediated ISOinduction of G0–G1 growth arrest andinhibition of anchorage-independentgrowth of bladder cancer cells. A,protein expression of Sp1, Sp4, andCyclin D1 in T24T and UMUC3 cells wasdetermined by Western blotting aftercells were treated with 10 mmol/L ISOfor indicated time periods. B, ISOtreatment did not inhibit ectopicexpressed GFP-Sp1, which reversed ISOattenuation of Cyclin D1 proteinexpression in T24T and UMUC3 cells.Ectopic expression of GFP-Sp1 reversedISO induction of G0–G1 growth arrest (Dand E) and inhibition of anchorage-independent growth (C and F) in T24Tand UMUC3 cells.






DNA (2 mg) was treated with sodium bisulfite using the EpiTectBisulfite Kit (Qiagen). Methylation-specific PCR was performedusing 20 ng of bisulfite-converted DNA and the specific primers.Methylated primer and unmethylated primers for miR-137 pro-moter were designed according to Shimizu and colleagues (27).PCR products were run on 2% agarose gel and visualized afterethidium bromide staining. Bisulfite-converted methylated andunmethylated DNA from EpiTect PCR Control DNA Set (Qiagen)was used as positive and negative controls.

Statistical analysesThe Student t test was used to determine the significance of

differences between different groups. The differences were con-sidered to be significant at P� 0.05. The Spearman correlation testwas chosen for examining the correlations between Sp1 expres-sion and Cyclin D1 expression and tumor weight.

ResultsISO represses tumor growth via downregulation of Sp1 proteinexpression both in vivo and in vitro

Our previous in vitro studies have well demonstrated that ISOtreatment induces cell-cycle G0–G1 arrest and inhibits cancer cellanchorage-independent growth through targeting Sp1/Cyclin D1axis in bladder cancer cells (4). To further determine the potentanticancer activities of ISO in vivo, we established a xenograftmodel in mice using human bladder cancer T24T cells, and thentreated these mice with ISO. As shown in Fig. 1A and B, ISOtreatment resulted in a dramatic inhibition of T24T xenografttumor growth as compared with vehicle control group (P < 0.01,n ¼ 6). ISO treatment also impaired the expression of Sp1 andCyclin D1 in the tumor tissues obtained from the tumor-bearingmice (Fig. 1C–E), which is consistent with our prior report in vitro(4). In addition, quantitative analysis of Sp1 expression intensityshowed that Sp1was positively associated with tumor weight (r¼0.825, P < 0.01) and Cyclin D1 expression (r¼ 0.916, P < 0.01) intumor tissues obtained from xenograft nudemice (Fig. 1F andG).In combination with our previous report, these findings stronglysuggest that downregulation of Sp1 protein is an important eventresponsible for anticancer activities of ISO. To further test thisnotion, we used human bladder cancer T24T and UMUC3 cells toestablish stable transfectants with GFP-Sp1 or scramble controlvector, respectively. As expected, ISO treatment significantly sup-pressed the expression of endogenous Sp1, but not endogenousSp4, accompanied by a marked decrease in Cyclin D1 expressionin a time-dependent manner (Fig. 2A). However, ISO treatmenthad no observable effects on exogenous expression of GFP-Sp1,ectopic expression of which attenuated the suppressive effectsof ISO treatment on Cyclin D1 expression in T24T and UMUC3(Fig. 2B). More interestingly, ectopic expression of Sp1 pro-nounced abolished the G0–G1 phase arrest and anchorage-inde-pendent growth inhibition in T24T and UMUC3 cells upon ISOtreatment (Fig. 2C–F). Taken together, these findings not onlyshowed that anticancer activities of ISO both in vitro and in vivowere associated with the downregulation of Sp1 and Cyclin D1 inhuman bladder cancer cells, but also emphasized the crucial rolefor Sp1 downregulation in ISO anticancer effects.

ISO inhibits Sp1 protein translationBecause ISO treatment specifically inhibited endogenous Sp1

expressionwithout altering ectopicGFP-Sp1protein expression in

T24T and UMUC3 cells, we anticipated that the downregulationof Sp1 expression by ISO treatment might occur at levels oftranscriptional, posttranscriptional, or translational. We, there-fore, examined the potential effects of ISO on Sp1 mRNA expres-sion. The results indicated that ISO treatment did not have anyobservable effects on Sp1mRNA expression level, thereby exclud-ing the possibility that ISO treatmentmodulates Sp1 expression attranscriptional or posttranscriptional levels (Fig. 3A). To furthertest the notion that ISO treatment might affect Sp1 expression attranslational level, we determined the effects of ISO on new Sp1protein synthesis using short-term 35S-methionine/cysteinepulse-labeling assay in T24T cells following ISO treatment. Asexpected, the incorporation of 35S-methionine/cysteine into new-ly synthesized Sp1 protein was gradually elevated along with theincubation time periods in T24T cells, whereas synthesis rate ofnew Sp1 protein was markedly attenuated in T24T cells that weretreated with ISO (Fig. 3B). This result demonstrates that ISOtreatment inhibits Sp1 protein translation.

miR-137 induction was crucial for inhibiting Sp1 proteintranslation by binding to the 30-UTR of Sp1

A large number of regulatory elements in either 30-UTR or50-UTR of mRNA have been identified and characterized fortheir regulation of protein translation (28). To elucidate themechanisms leading to ISO inhibition of Sp1 protein trans-lation, Sp1 30-UTR luciferase reporter was transiently cotrans-fected with pRL-TK into T24T and UMUC3 cells, respectively.The transfectants were used to evaluate the effect of ISO on 30-UTR luciferase reporter activity. As shown in Fig. 4A, ISO treat-ment resulted in a dramatic reduction of Sp1 30-UTR activity in atime-dependent manner in both T24T and UMUC3 cells, indi-cating that Sp1 mRNA 30-UTR might be regulated by ISO for itsinhibition of Sp1 protein translation.MiRNAs have been reportedto bind to mRNA 30-UTR and suppress protein translation (29).Therefore, we used the miRcode, miRWalk, and TargetScan data-base to screen the possible miRNAs that could potentially target

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Figure 3.ISO treatment specifically suppressed Sp1 protein translation. A, total RNAisolated from the T24T cells treated with 10 mmol/L ISO for indicated timepoints, and then subjected to RT-PCR for the determination of Sp1 mRNAexpression level. The b-actinwas used as a loading control. B, T24T cells weretreated with 10 mmol/L ISO, and newly synthesized Sp1 protein wasmonitored by pulse assay using 35S-labeled methionine/cysteine; WCL,whole cell lysate. Coomassie blue staining was used for protein loadingcontrol as described in the Materials and Methods section.

Zeng et al.





Sp1 mRNA 30-UTR. The results obtained from comprehensiveanalysis indicated thatmiR-29a,miR-29b,miR-29c,miR-137, andmiR-145 could have potential binding to 30-UTR of Sp1 mRNA(Fig. 4B). To identify which of these miRNAs was responsible forregulation of Sp1 protein translation, we evaluated thesemiRNA'sexpression in both cell lines treated with ISO. These data showedthat ISO treatment induced the expression of miR-145 and miR-137, without affecting the others in both T24T and UMUC3 cells(Fig. 4C and D), indicating that miR-137 and miR-145 might beinvolved in downregulation of Sp1 protein translation followedISO treatment.

To test whether miR-137 and/or miR-145 played a role inregulation of Sp1 protein translation, miR-145 and miR-137were stably transfected into T24T cells, respectively, and theectopic expression levels of miR-145 and miR-137 were eval-uated by real-time PCR as shown in Fig. 5A and B. Overexpres-sion of miR-137 blocked Sp1 30-UTR luciferase reporter activityin a dual-luciferase reporter assay, whereas ectopic expression ofmiR-145 did not show the observable effect under same exper-imental conditions (Fig. 5C). Consistently, stable overexpres-

sion of miR-137 in UMUC3 also dramatically decreased the Sp130-UTR luciferase reporter activity (Fig. 5D and E). To definewhether miR-137 inhibition was due to its specific binding topotential miR-137 binding site at Sp1 mRNA 30-UTR, we con-structed mutant of Sp1 30-UTR luciferase reporter as displayedin Fig. 5F. BothWT andmutant of Sp1 30-UTR luciferase reporterwere stably transfected into T24T (vector) and T24T (miR-137)transfectants, respectively. As shown in Fig. 5G, miR-137 over-expression significantly reduced WT Sp1 30-UTR luciferasereporter activity, whereas mutation of miR-137 binding site atSp1 30-UTR luciferase reporter completely attenuated miR-137inhibition of Sp1 30-UTR luciferase reporter activity, indicatingthat miR-137 is likely to bind to Sp1 30-UTR directly andregulate Sp1 protein translation. Consistent with miR-137inhibition of Sp1 30-UTR luciferase reporter activity, overexpres-sion of miR-137 also impaired Sp1 and Cyclin D1 proteinexpression in both T24T and UMUC3 cells (Fig. 5H), and itdid not show any inhibitory effect on exogenous GFP-Sp1protein expression and GFP-Sp1–mediated Cyclin D1 expres-sion (Fig. 5I).

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Figure 4.ISO treatment inhibited Sp1 mRNA30-UTR activity and induced theexpression of miR-137 and miR-145.A, Sp1 30-UTR luciferase reporter wastransfected into T24T and UMUC3cells, and the transfectants weretreated with ISO (10 mmol/L) forindicated time points. The cells werethen extracted for determination ofluciferase activity. B, the potentialmiRNA binding sites in Sp1 mRNA30-UTR predicted by the miRcode,miRWalk, and TargetScan database.C and D, the relative expression levelsof miRNAs were evaluated byquantitative real-time PCR in T24T (C)and UMUC3 cells (D) followed by ISO(10 mmol/L) treatment at the indicatedtime periods.






miR-137 was downregulated in human bladder cancer tissues,and ectopic expression of miR-137 suppressed bladder cancercell monolayer growth, anchorage-independent growth, andinducedG0–G1 cell growth arrest in humanbladder cancer cells

miR-137 gene locates on chromosome 1p22 and has beenreported to be downregulated in some cancer tissues, such asbreast cancer (30), colorectal cancer (31), andnon–small cell lungcancer (32). However, the association of miR-137 with humanbladder cancer has not been reported yet to the best of ourknowledge. To explore this possibility, the expression level ofmiR-137 in bladder cancer tissues was determined and comparedwith that in the paired adjacent nontumorous bladder tissues. Theresults indicated that miR-137 expression was almost completelyimpaired in human bladder cancer tissues as compared with thatin adjacent normal bladder tissues (Fig. 6A, n¼ 26). To assess thebiologic role of miR-137 in regulation of human bladder cancercell growth, we stably transfectedmiR-137 into T24T cells, and theeffect of miR-137 overexpression on monolayer growth, anchor-age-independent growth, and cell cycles was evaluated in com-parison with scramble vector transfectants. As shown in Fig. 6B,overexpression of miR-137 could mimic ISO treatment andreduce bladder cancer monolayer growth. Furthermore, overex-pression of miR-137 also profoundly inducted G0–G1 growtharrest accompanied with attenuation of anchorage-independentgrowth in T24T cells, and these biologic effects of miR-137 couldbe reversed by ectopic expression of GFP-Sp1 (Fig. 6C–E).

Above results from in vitro human bladder cancer cellsdemonstrate that miR-137 is induced by ISO treatment, which

was crucial for ISO inhibition of Sp1 protein translation bybinding to Sp1 mRNA 30-UTR. We next evaluated the ISO effecton miR-137 expression in mouse tumor nodules obtained fromin vivo animal studies. The results revealed that miR-137expression was significantly increased in tumor nodules frommice that were treated with ISO in comparison with these thatwere treated with control vehicle (Fig. 7A). To provide a directevidence showing the critical role of miR-137 in ISO inhibitionof Sp1 and Cyclin D1 expression as well as in ISO anticanceractivity, the specific miR-137 inhibitor was stably transfectedinto T24T cells, and the stable transfectants were used toevaluate its role in miR-137 induction by ISO in its inhibitionof Sp1 protein expression, Sp1 mRNA 30-UTR activity, CyclinD1 expression, anchorage-independent growth, as well as theinduction of G0–G1 growth arrest in human bladder cancercells. As illustrated in Fig. 7B, miR-137 inhibitor expression didattenuate miR-137 induction followed by ISO treatment. Con-sistently, the ectopic expression of miR-137 inhibitor alsoreversed ISO downregulation of Sp1 30-UTR activity, Sp1 pro-tein expression as well as Cyclin D1 protein expression (Fig. 7Cand D). Moreover, the inhibition of ISO-induced miR-137expression by miR-137 inhibitor also attenuated ISO inductionof G0–G1 cell growth arrest and ISO inhibition of anchorage-independent growth in human bladder cancer T24T cells(Fig. 7E and F). Collectively, our results clearly demonstratethat ISO-induced miR-137 expression acted as a tumor sup-pressor by binding to Sp1 mRNA 30-UTR and inhibiting Sp1protein translation, by which Cyclin D1 attenuates expression,

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Zeng et al.





subsequently resulting in cell growth arrest and anchorage-independent growth inhibition in the bladder cancer cells.

DiscussionTo acquire more evidences for further translational applica-

tion of ISO in the management of clinical patients, the in vivoanimal verification and extensive mechanistic in vitro studieswere carried on in current study. First, the in vivo animal studiesdemonstrated that the antitumor activity of ISO in the subcu-taneously transplanted tumor of human bladder cancer in nudemouse model was in line with our previous in vitro studies.Second, we consistently highlighted a crucial role of ISO down-regulation of Sp1 protein expression as a key factor mediatingits anticancer activity both in vivo and in vitro. Our extensive invitro studies revealed that the anticancer effect of ISO wasmediated by its downregulation of Sp1 protein translation viainduction of miR-137, which directly binds to Sp1 mRNA 30-UTR region. Although miR-137 expression is reported in a fewtypes of tumors, including colorectal (33), gastric (34), lung(32), and glioblastoma (35), its expression and function inhuman bladder cancers have not been explored yet to the bestof our knowledge. Our studies indicated that miR-137 expres-sion was impaired in human bladder cancer tissues, and it actedas a tumor-suppressive miRNA that suppresses the anchorage-independent growth and induces cell-cycle G0–G1 arrest inhuman bladder cancer cells.

Chinese herb G. cleistostachyum has been used as a traditionalChinese medicine for treatment for arthritis, bronchitis, car-diovascular system disease, and several cancers including blad-der cancer almost for a century (36). ISO, a new derivative ofstilbene compound, isolated from G. cleistostachyum and itschemical structure is a 4-methoxyresveratrol (37). Our most

recent studies have explored anticancer activity of ISO byinducing cell-cycle G0–G1 arrest and inhibiting cancer cellanchorage-independent growth through downregulating Sp1/Cyclin D1 axis in vitro human bladder cancer cells, suggestingthat ISO has a potential being a novel mechanism-based cancertherapeutic agent against human bladder cancer in vitro. Thisprovides a basis for possible clinical utilization of ISO as apreventive and therapeutic agent against bladder cancers inclinical patients (4). However, the research lacked in vivoanimal verification and extensive in vitro studies. In presentstudies, the xenograft nude mouse model was used for theintensive studies on the anticancer effect of ISO in bladdercancers. Consistent with the findings in vitro, our new resultsobtained from current studies revealed that ISO is a potentagent for its inhibition of the tumor growth in the xenograftnude mouse model. We also observed that Sp1 and its regu-lated Cyclin D1 expression were also downregulated in trans-planted tumor nodules in mice followed with ISO treatment.The further analysis revealed that Sp1 expression, Cyclin D1expression, and tumor growth were very well positively corre-lated. Current in vivo animal studies together with our early invitro studies demonstrate that ISO is a novel mechanism-basedcancer therapeutic agent that mainly targets Sp1/Cyclin D1 axisin human bladder cancers.

Sp1 is an important transcription factor that is involved in theregulation of many gene expressions and cellular functions (6)and is overexpressed in various cancer cell lines and tumor tissues(9–15). Sp1-regulated genes and oncogenes play an importantrole in cancer cell proliferation, survival, angiogenesis, andinflammation (38–41). The transcription factor is ideal for devel-opment of mechanism-based drugs because Sp1 expression isassociated with aging (42–44). Several drugs that target Sp1 havebeen identified, and these include the NSAIDs tolfenamic acid,

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Figure 6.Downregulation of miR-137 in humanbladder cancer tissues and miR-137overexpression suppressedanchorage-independent growth andinduced G0–G1 growth arrest of humanbladder cancer cells. A, the relativeexpression levels of miR-137 in bladdercancer tissues and normal tissuesdetermined by quantitative real-timePCR. Expression was shown as a log2(miR-137/U6) change. B–D,overexpressed miR-137 in T24T cellsinhibited monolayer growth (B) andinduced G0–G1 growth arrest (C) andanchorage-independent growth (D). E,ectopic expression of GFP-Sp1reversed the inhibition of the inductionof cell-cycle arrest (C) and anchorage-independent growth (E) caused bymiR-137 overexpression in T24T cells.






COX-2 inhibitors, and the nitro-NSAID GT-094, and severalnatural products, including betulinic acid (BA), celastrol, and thesynthetic triterpenoidsmethyl 2-cyano-3, 12-dioxooleana-a-dien-28-oate (CDDO-Me) and methyl,2-cyano-3,4-dioxo-18b-olean-1,12-dien- 30-oate (CDODA-Me; refs. 41, 45–47). Our previousstudies reveal that Sp1 downregulation is essential for ISO anti-cancer effect on human bladder cancer cells. However, themechanisms underlying ISO downregulation of Sp1 were stillunknown. It is reported that clinically used andmechanism-basedanticancer drugs downregulate Sp1 proteins in cancer cell linesthrough multiple pathways that are dependent on the drug andcell context (48). For example, curcumin induces proteasome-dependent downregulation of Sp1 in bladder cancer cells (40),whereas in pancreatic cancer cells, the effects of curcumin ondecreased expression of Sp1 are ROS dependent (39, 48). Inpancreatic cancer cells, tolfenamic acid induced degradation ofSp1 (49), but differently, curcumin induced ROS-dependentdownregulation of Sp1 (39, 48). Our current studies demonstrat-ed that ISO treatment specifically inhibited Sp1 protein transla-tion without affecting sp1 mRNA level via its induction of miR-137. MiRNA, approximately 22 nucleotides noncoding RNAs,has been reported to be negatively regulator mediating geneexpression by modulating mRNA stability or suppressing proteintranslation by binding to its targeting mRNA 30-UTR (29). It isestimated that the expression of at least 30% of human genes is

regulated by miRNAs (50). For example, miR-29b inhibitsSp1 expression in tongue squamous cell carcinoma (51). There-fore, we speculated that miRNAs might involve in the ISO down-regulation of Sp1 expression. The induction ofmiRNAs, includingmiR-145 and miR-137, was observed in T24T and UMUC3cells treated with ISO. Ectopic expression of miR-137, but notmiR-145, showed suppression of Sp1 mRNA 30-UTR activityand protein expression in human bladder cancer T24T andUMUC3 cells, whereas mutation of miR-137 binding site in Sp1mRNA 30-UTR luciferase reporter attenuated miR-137 inhibitionof Sp1mRNA30-UTR activity, indicating thatmiR-137 specificallytargets Sp1 mRNA 30-UTR for its inhibition of Sp1 proteintranslation, further revealing the identification of Sp1 being anovel miR-137–targeted gene.

miR-137 is located on human chromosome 1p22 and has beenimplicated to act as a tumor suppressor in several cancer types.Increasing numbers of miR-137 target genes have been documen-ted and have shown to play important roles in various humancancers. For example, Liu and colleagues report that miR-137regulates epithelial-mesenchymal transition (EMT) and inhibitscell migration via downregulation of Twist1 in gastrointestinalstromal tumor (52). Chen and colleagues demonstrate that miR-137 suppresses tumor progression and metastasis in colorectalcancer (53). Liu and colleagues recently also report that miR-137suppresses tumor growth andmetastasis in humanhepatocellular

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Figure 7.miR-137 inhibitor reversed ISO inhibition of Sp1 and Cyclin D1 protein expression, Sp1 30-UTR activity, and anchorage-independent growth, as well as abolished ISOinduction of G0–G1 growth arrest in bladder cancer cells. A, ISO treatment induced miR-137 expression in tumor nodules from mice (n ¼ 6). B, miR-137inhibitor inhibited induction ofmiR-137 by ISO treatment in T24Tcells. C,miR-137 inhibitor reversed ISO inhibitionof Sp1 andCyclin D1 protein expression in T24T cells.D, miR-137 binding site was crucial for ISO inhibition of Sp1 30-UTR activity. E and F, miR-137 inhibitor reversed ISO induction of G0–G1 growth arrest and inhibitionof anchorage-independent growth of T24T cells. G, ISO treatment did not affect miR-137 promoter methylation. H, the proposed mechanism underlying ISOanticancer effect.

Zeng et al.





carcinoma by targeting AKT2 (54). The studies from Shimizu andcolleagues showed that ectopic expression of miR-137 suppressesbladder cancer cell proliferation (27), whereas another studiesindicated that overexpression of miR-137 promotes cell prolifer-ation, migration, and invasion of bladder cancer cells (55). In ourstudies presented here, we found thatmiR-137 expression level inprimary human bladder cancer tissues was dramatically down-regulated as compared with their paired adjacent nontumoroustissues. Further intensive in vitro studies displayed that miR-137induction was able to induce G0–G1 cell growth arrest andsuppress monolayer cancer cell growth and anchorage-indepen-dent growth in bladder cancer cell lines via inhibiting Sp1/CyclinD1 axis. It is reported that miR-137 promoter region is frequentlymethylated inprimarybladder tumors than innormal urothelium(27). Thus, we propose that miR-137 downregulation in humanbladder cancer tissuesmight be due to the hypermethylation of itspromoter region, while induction of miR-137 in human bladdercancer cells upon ISO treatment might be associated with reduc-tion of miR-137 promoter methylation followed by ISO treat-ment. However, the results obtained from methylation-specificPCR showed thatmiR-137promotermethylationwasnot affectedupon ISO treatment (Fig. 7G), revealing that ISO-induced miR-137 expression was through miR-137 promoter methylation-independent manner. Further investigation of the mechanismsunderlying ISOupregulation ofmiR-137will be highly significantfor providing deep insight into understanding anticancer effect ofISO, and is now ongoing project in our research program.

In summary, our studies demonstrated that ISO exhibitedanticancer effect on human bladder cancer experimental systemboth in vivo and in vitro via its upregulation ofmiR-137 expression,which in turn inhibited Sp1 protein translation and subsequentlyresulted in Cyclin D1 protein expression, leading to cell-cycleG0–G1 arrest and inhibiting anchorage-independent cell growthof human bladder cancer cells as illustrated in Fig. 7H. Thecomprehensive studies including in vivo and in vitro studies are

crucial for potential translational application of ISO in the man-agement of clinical patients. Our studies not only provide a novelinsight into understanding the anticancer activity of ISO, but alsoreveal that ISOcould beused as a therapeutic drug for treatment ofhuman bladder cancer with miR-137 downregulation.

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

Authors' ContributionsConception and design: H. Jin, C. HuangDevelopment of methodology: Z. Xu, J. Li, G. JiangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Zeng, J. Gu, H. Huang, G. Gao, X. Zhang, J. Li,G. Jiang, H. SunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Zeng, Z. Xu, J. Gu, H. Huang, C. HuangWriting, review, and/or revision of the manuscript: X. Zeng, H. Jin, C. HuangStudy supervision: H. Huang, C. Huang

AcknowledgmentsThe authors thank Dr. Guido Marcucci from the Department of Medicine,

Ohio State University, for the gift of human Sp1 30-UTR luciferase reporter andDr. Renato Baserga from the Department of Cancer Biology, Thomas JeffersonUniversity, for the gift of miR-145 expression construct pBluescript-miR-145.

Grant SupportThis work was partially supported by grants from NIH/NCI [CA112557,

CA177665, and CA165980 (to C. Huang)], NIH/NIEHS ES000260 (toC. Huang), and NSFC 81229002 (to C. Huang) as well as Key Project of Scienceand Technology Innovation Team of Zhejiang Province (2013TD10; toH. Huang).

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

Received July 23, 2015; revised December 3, 2015; accepted December 30,2015; published OnlineFirst February 1, 2016.

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Zeng et al.




2016;15:512-522. Published OnlineFirst February 1, 2016.Mol Cancer Ther Xingruo Zeng, Zhou Xu, Jiayan Gu, et al.

In Vivo and In VitroSp1 Protein Translation and Mediates Its Anticancer Activity Both Induction of miR-137 by Isorhapontigenin (ISO) Directly Targets

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