MicroRNA-145 Targets the Metalloprotease ADAM17 and Is Suppressed in Renal Cell Carcinoma Patients

MicroRNA-145 Targets theMetalloprotease ADAM17and Is Suppressed in RenalCell Carcinoma Patients1

Kai Doberstein*, Nico Steinmeyer*,Ann-Kathrin Hartmetz*, Wolfgang Eberhardt*,Michel Mittelbronn†, Patrick N. Harter†,Eva Juengel‡, Roman Blaheta‡,Josef Pfeilschifter* and Paul Gutwein*

*Pharmazentrum Frankfurt/Zentrum für Arzneimittelforschung,Entwicklung und Sicherheit (ZAFES), Goethe UniversityHospital, Frankfurt am Main, Germany; †Edinger Institute(Institute of Neurology), Goethe University, Frankfurtam Main, Germany; ‡Department of Urology, GoetheUniversity Hospital, Frankfurt am Main, Germany

AbstractA disintegrin and metalloproteinase 17 (ADAM17) is a metalloprotease that is overexpressed in many cancer types,including renal cancers. However, the regulatorymechanisms of ADAM17 in cancer development and progression arepoorly understood. In the present work, we provide evidence using overexpression and inhibition of microRNA 145(miR-145) that miR-145 negatively regulates ADAM17 expression. Furthermore, we show that ADAM17 negativelyregulates miR-145 through tumor necrosis factor–α, resulting in a reciprocal negative feedback loop. In this study,the expression of ADAM17 and miR-145 correlated negatively in renal cancer tumor tissues and cell lines, suggestingan important regulatory mechanism. Additionally, we showed that the regulation of ADAM17 is partly involved in theeffects of miR-145 on proliferation and migration, whereas no involvement in chemosensitivity was observed. Im-portantly, in the healthy kidney, miR-145 was detected in different cell types including tubular cells, which are consid-ered the origin of renal cancer. In renal cancer cell lines, miR-145 expression was strongly suppressed bymethylation.In summary, miR-145 is downregulated in renal cancer patients, which leads to the up-regulation of ADAM17 in renalcancer. Importantly, miR-145 and ADAM17 are regulated in a reciprocal negative feedback loop.

Neoplasia (2013) 15, 218–230

IntroductionRenal cell carcinoma (RCC) originates from renal tubules and is themost common cancer of the kidney, accounting for 90% to 95% ofall renal tumors in adults [1]. Importantly, 60% of all affected patientsdevelop metastases. Only 10% of RCC patients with metastases survivelonger than 5 years, whereas 80% of RCC patients with non-metastaticdisease survive 5 years or longer [2]. Given the scarce number of thera-peutic regimens, alternative approaches are urgently needed to prolongpatient survival.An interesting potential target molecule is the metalloprotease a dis-

integrin and metalloproteinase 17 (ADAM17), which is overexpressedin several types of cancer, including renal cancer [3–11]. ADAM17was originally identified as the protease of the tumor necrosis factor–α(TNF-α), which is important during inflammation and for the cancermicroenvironment [12,13].

In RCC, ADAM17 expression increases with the degree of malig-nancy and it is essential to form xenograft tumors [11,14]. Importantly,more than 70 cellular substrates have been identified for ADAM17,including TNF-α, transforming growth factor–α, and epidermal growth

Abbreviations: ADAM, A disintegrin and metalloproteinase; RCC, renal cell carcinoma;TNF, tumor necrosis factorAddress all correspondence to: Kai Doberstein, MSc, Pharmazentrum Frankfurt/Zentrumfür Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES), Goethe UniversityHospital, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.E-mail: [email protected] article refers to supplementary materials, which are designated by Figures W1 toW3 and are available online at www.neoplasia.com.Received 27 July 2012; Revised 21 December 2012; Accepted 27 December 2012

Copyright © 2013 Neoplasia Press, Inc. All rights reserved 1522-8002/13/$25.00DOI 10.1593/neo.121222

www.neoplasia.com

Volume 15 Number 2 February 2013 pp. 218–230 218

factor receptor (EGFR) ligands [15]. Interestingly, the expression levelsof members of the ADAM family can be regulated by small non-codingmicroRNAs (miRNAs) [16–18]. Furthermore, miRNAs were shown tohave an important role in the regulation of gene networks, includingthose that are essential for cancer development [19–21].miRNA 145 (miR-145) plays a crucial role in the differentiation of

smooth muscle cells (SMCs) [22–24]. miR-145 is downregulated inmany tumors, including hematopoietic tumors and solid tumors of thebreast, ovary, head andneck, prostate, skin, lung, bladder, and cervix [25–28]. Furthermore, the ectopic expression ofmiR-145 in cancer cells led toa loss in cell viability and induced cell death [26,29,30]. It has been con-sidered that the down-regulation of miR-145 is an early event in manycancers, suggesting an important role in several cancer-related pathways[31,32]. A number of genes that are important in different cancer path-ways have been identified and validated as target genes of miR-145,including plasminogen activator inhibitor-1 (PAI-1), octamer-bindingtranscription factor 4 (Oct-4), sex determining region Y (SRY)-box 2(Sox-2), c-Myc, and p70S6K1 [33–36]. miR-145 is downregulated bypromoter hypermethylation in many cancers [37–39]. Additionally, thetumor suppressor gene p53, which is inactive in about 50% of all can-cers, upregulates miR-145 expression, whereas, in contrast, the Rasoncogene represses miR-145 expression [37,40]. It has been implicatedby Gregersen et al. that ADAM17 may be a target of miR-145 [41].In this study, we identified miR-145 as an miRNA that is down-

regulated in the majority of renal cell cancers by methylation. Further-more, we provide evidence that miR-145 decreases proliferation andinvasion by directly targeting the 3′ untranslated region (3′UTR) ofADAM17 mRNA. Additionally, we show that ADAM17 negativelyregulates miR-145 expression through TNF-α, leading to a reciprocalnegative feedback loop. Importantly, we found a negative correlationof miR-145 and ADAM17 expression in renal cancer samples, sug-gesting that these molecules could be promising targets for furthertreatment approaches.

Materials and Methods

Substances and AntibodiesTo detect ADAM17 protein expression, we used the polyclonal

antibody from Santa Cruz Biotechnology (Heidelberg, Germany;sc-13973). A β-actin antibody from Sigma-Aldrich (Taufkirchen,Germany) was used as a loading control. The metalloprotease inhibitorsTAPI-0 and TAPI-2 were obtained from Calbiochem (Darmstadt,Germany). Recombinant human TNF-α was purchased from Peprotech(London, United Kingdom). Phorbol 12-myristate 13-acetate (PMA)and 5-aza-2′-deoxycytidine (AZA) were obtained from Sigma (St Louis,MO) and cisplatin from Teva (Radebeul, Germany).

Cell CultureThe renal carcinoma cell line A498 was obtained from Dr K.

Joehrer (Innsbruck Medical University, Innsbruck, Austria), whereasthe renal carcinoma cell line Foehn was a kind gift from Prof. Altevogt(German Cancer Research Center, Heidelberg, Germany). The renalcarcinoma cell lines RCC4 and 786-0 were obtained from Prof. Krek(ETH Zurich, Zürich, Switzerland). Protein isolation and Westernblot analyses were performed as previously described [42].

cDNA Synthesis and Polymerase Chain Reaction AnalysisRNA from cultured cells was isolated using the RNA Easy Kit accord-

ing to the manufacturer’s protocol (Qiagen, Hilden, Germany). Equal

amounts of total cellular RNA (1 μg) were reverse transcribed with ran-dom primer by the use of M-MuLV Reverse Transcriptase (Fermentas,St Leon-Rot, Germany). Transcribed cDNAs were used for polymerasechain reaction (PCR) with specific primers for ADAM17 (435 bp,5′-GCATTCTCAAGTCTCCACAAG-3′ and 5′-CCTCATTCGGG-GCACATTCTG-3′), PAI-1 (183 bp, 5′-GAGGTGCCTCTCTCTG-CCCTCACCAACATT-3′ and 5′-AGCCTGAAACTGTCTGAACAT-GTCG-3′), c-Myc (478 bp, 5′-TACCCTCTCAACGACAGCAG-3′and 5′-TCTTGACATTCTCCTCGGTG-3′), β-actin (234 bp, 5′-GGACTTCGAGCAAGAGATGG-3′ and 5′-AGCACTGTGTTGG-CGTACAG-3′), and pri-miR-145 (73 bp, 5′-TGGATTTGCCTCCT-TCCCA-3′ and 5′-TTGAACCCTCATCCT GTGAGCC-3′). Themethod described by Chen et al. was followed for stem-loop reverse tran-scription of mature miRNAs [43]. For miR-145, the stem-loop primer5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTG-GATACGACAGGGAT-3′ was used, and for U48, the stem-loopprimer 5′-GTTGGCTCTGGTGCAGGGTCCGAGGT-ATTCGC-ACCAGAGCCAACGGTCAG-3′ was used. For miR-143, the com-mercially available primers from Exiqon (Vedbaek, Denmark) wereused (#204190). The reverse transcribed product of miR-145 was thenamplified by PCR with specific primers (5′-CGGCGTCCAGTTTT-CCCAGG-3′ and 5′-GTGCAGGGTCCGAGGT-3′, and for U48,5′-CGACGAGTGATGATGAC-3′ and 5′-GTGCAGGGTCCG-AGGT-3′).

Real-time PCRReal-time PCR was performed as described in the protocol from

the Absolute Blue QPCR SYBR Green Low ROX Kit (ThermoScientific, Hilden, Germany). The same primers as for the conven-tional PCR were also used for the real-time PCR. The CT values ofanalyzed RNA levels were normalized to the CT values of β-actin formRNA and for miR-145 to U48 (housekeeping miRNA) within thesame sample.

Luciferase ConstructsTo construct luciferase reporter plasmids, we inserted various target

fragments into multiple cloning site (MCS; XhoI and NotI) down-stream of the Renilla luciferase reporter gene in the psiCHECK-2promoter vector (Promega, Madison, WI). 5′ Modified primers carry-ing restriction sites for either XhoI or NotI were used (sequence printedin bold). The sequence from 3075 to 3082 (5′-AACTGGAA-3′) inthe human ADAM17 mRNA (NM_003183.4) was termed miR-145binding site.The 761-nt fragment of the ADAM17 3′UTR (2613-3373, NM_

003183.4) was generated using the following primer set: forward,5′-atactcgagACTGCAGCGTCAGAATCGTGTTGA-3′ and reverse,5′-atagcggccgcCCCAGCAAATCAGGGCCCTAAACA-3′.Site-directed mutagenesis of the miR-145 binding site in the

ADAM17 3′UTRwas carried out with the QuickChange Site-DirectedMutagenesis Kit (Stratagene, La Jolla, CA), using the followingprimers: 5′-GAGGCATTTGGCATTTATTTGTGATGACAgcaga-aATAGTTTTTTT-3′ and 5′-AAAAAAACTATttctgcTGTCATCAC-AAATAAATGCCAAATGCCTC-3′.A fragment containing the perfect matching sequence with the ma-

ture miR-145, 5′-atactcgagAGGGATTCCTGGGAAAACTGGAC-gcggccgcata-3′ (the matching sequence is underlined), was cloned(psiCHECK-2-p145). All constructs were sequenced.

Neoplasia Vol. 15, No. 2, 2013 miR-145 Regulates ADAM17 in Renal Cell Carcinoma Doberstein et al. 219

Luciferase AssayHuman embryonic kidney 293 (HEK293) cells (3 × 105 per six-well

plate) were transfected using the Lipofectamine 2000 transfectionreagent (Invitrogen, Carlsbad, CA). Control RNA (20 nM) or miR-145 was co-transfected with 400 ng of appropriate plasmids and lysedfor luciferase assay 24 hours after transfection. Luciferase assays were per-formed using a Dual-Luciferase Reporter Assay System (Promega)according to the manufacturer’s protocol. Firefly luciferase was usedfor normalization.

Oligonucleotide TransfectionThe following siRNA duplexes (MWG Biotech AG, Ebersberg,

Germany) were used for down-regulation of the corresponding proteinexpression: ADAM17-siRNA, 5′-GAGAAGCUUGAUUCUUUG-CTT-3′. The transfection of miR-145 mimic (10 nM), miR-145 inhib-itor (10 nM; both Qiagen), and miR-143 locked nucleic acid (LNA;Exiqon) inhibitors was performed in the same manner. As negativecontrols, unspecific AllStars negative control RNA from Qiagen orLNA control from Exiqon was used. Twenty-four hours before transfec-tion, 0.4 × 105 cells were seeded in six-well plates. Transfection of siRNAwas carried out using Oligofectamine (Invitrogen, Karlsruhe, Germany)together with 10 nM siRNA duplex per well as previously described [44].

Immunohistochemistry and In Situ HybridizationThe in situ hybridization was performed with a 3′ and 5′ double DIG-

labeled LNA miR-145 probe (Exiqon). The hybridization protocol hasbeen described in detail by Jorgensen et al. [45]. After miR-145 staining,development and immunohistochemistry staining, starting with the firstantibody, was continued with the same slide. Immunohistochemistryhas been previously described [46].

Cell Cycle AnalysisSeventy-two hours after transfection, cells were trypsinized, washed

in phosphate-buffered saline, and fixed with 70% ethanol at −20°C.After centrifugation, cells were incubated in hypotonic solution con-taining 50 μg/ml propidium iodide, 0.1% sodium citrate, 0.1% TritonX-100, and 20 μg/ml DNase-free RNaseA for 30 minutes at 37°C.Finally, cells were analyzed using flow cytometry in a linear mode. Eachassay was performed in triplicates and repeated at least three times.

Patient MaterialTumor specimens and adjacent normal kidney tissues were col-

lected from a total of 15 patients undergoing nephrectomy for RCC.All specimens were obtained on the basis of their availability forresearch purpose and under a protocol approved by the local medicalethics committee of Goethe University Frankfurt. Written consent wasobtained from the patients in the study.

Cell Invasion AssayThe cell migration assay has been described earlier [47].

Proliferation AssayThe proliferation assay has been previously described [48].

Statistical AnalysisIf not otherwise indicated in the figure legends, Student’s t test was

used for statistical analyses.

Results

miR-145 Downregulates ADAM17 Protein Expression inRenal Cancer Cell LinesThe computational algorithms TargetScan, PicTar, and miRANDA

were used to identify miRNAs that could potentially regulate ADAM17expression [49–51]. These programs search for miRNAs that targetevolutionary conserved sequences in the 3′UTR of the mRNA of themolecule of interest. miR-145 was one of two miRNAs predicted byall three programs to bind to the ADAM17 3′UTR (Figure 1, A andB). Importantly, it was shown in three microarray studies by miRNAprofiling of renal cancer patients that miR-145 is regulated in cancertissue [52–54].Next, we investigated the miR-145 levels in a primary renal tubular

cell line and in four renal cancer cell lines. The primary renal tubularcell line HRCepiC expressed higher levels of miR-145 than the renalcancer cell lines RCC4, 786-0, A498, and Foehn (Figure 1C ). Incontrast, the tubular cell line HRCepiC expressed the lowest amountsof ADAM17 protein and mRNA when compared to renal cancer celllines (Figure 1, D and E).To analyze the miR-145 and ADAM17 expression in renal cancer

patients, we isolated renal cell cancer and corresponding normal kidneyRNA from 15 patients and analyzed the expression levels by real-timePCR (Figure 1F). Interestingly, in only one tumor specimen, miR-145was moderately upregulated (+3.8); in four renal cancer tissues, it wasvirtually unchanged (+1.4 to −1.1), while in 10 renal cancer tissues,it was downregulated, ranging from 1.7-fold to more than 65-folddown-regulation, with an overall average of 8.9-fold down-regulation(P < 10−4). In contrast, only one patient displayed a decrease inADAM17 expression (1.9), whereas six displayed almost no change(+1.2 to −1.4) and eight showed an up-regulation (+1.6 to +5.8) intumor tissue compared to normal tissue with an overall average of1.2-fold increase (P < .05).The expression of miR-145 was further plotted versus the mRNA

expression of ADAM17 for every individual patient (Figure 1G). Inter-estingly, all patient values were plotted in a small corridor, with the ex-ception of patient number 218 that displayed the strongest decreasein miR-145 (−65.2) and ADAM17 (−1.9) expression. When analyzingthe values in the marked corridor (14 of 15 patients), miR-145 andADAM17 showed a significant negative correlation (K = −0.67), pro-viding evidence for the regulation of ADAM17 by miR-145 in vivo.In contrast, when analyzing the known genes c-Myc and PAI-1, whichhave been described as miR-145 target genes, we could not find acorrelation (Figure 1, H and I).To determine the localization of miR-145 in normal human renal

tissue, we performed in situ hybridization with miR-145–specificprobes, followed by immunohistochemical staining with an antibodyagainst aquaporin-1, which is known as a marker of proximal tubules[55]. miR-145 was strongly expressed in SMCs of blood vessels andinside the glomerulus (Figure 1, I , K , and N ). Additionally, tubulesadjacent to glomeruli and small tubules displayed distinct miR-145expression (Figure 1, M and N ).

miR-145 Regulates ADAM17 Expression through DirectBinding to the 3′UTRNext, we evaluated the ability to inhibit or overexpress miR-145 using

a commercial miR-145 inhibitor or mimic in the renal cancer cell linesA498 and Foehn. The cells were transfected with miR-145 inhibitor,the miR-145 mimic, or a control RNA. Transfection efficiency was

220 miR-145 Regulates ADAM17 in Renal Cell Carcinoma Doberstein et al. Neoplasia Vol. 15, No. 2, 2013

measured by reverse transcription–PCR, which demonstrated thatthe miR-145 levels were efficiently changed (Figure W1, A and B).The reduction of miR-145 after inhibitor transfection indicates a deg-radation of mature miR-145 [56]. Importantly, in A498 and Foehncells, the transfection of miR-145 mimic led to a strong reductionin ADAM17 protein expression, whereas the transfection with themiR-145 inhibitor increased ADAM17 protein expression in both celllines (Figure 2, A and B).To investigate whether miR-145 represses ADAM17 expression

through direct binding on the ADAM17 3′UTR, we introducedthe predicted miR-145 binding site into the 3′UTR of a luciferasereporter gene. We found that the transfection of HEK293 cells withmiR-145 mimics reduced the luciferase activity significantly, whereasit had no effects on the mutated binding site (Figure 2C ).

Taken together, we observed that miR-145 regulates the proteinexpression of ADAM17, which might be due to the direct interactionof miR-145 with the 3′UTR of ADAM17 mRNA.

Inhibition of miR-143 Reduces ADAM17 Expression inRenal Cancer CellsBecause miR-145 is transcribed in a cluster with miR-143, and

both have been shown to work synergistically in several cell models,we analyzed the expression of miR-143 in our renal cancer cell model.When comparing different renal cancer cell lines, the A498 cellline had the highest, whereas Foehn cells had the lowest expressionof miR-143 (Figure 3A). The transfection of the Foehn cells withmiR-145 mimic leads to an increase in miR-143 levels, whereas in the

Figure 1. miR-145 regulates ADAM17 expression in renal cancer cell line. (A) Predicted duplex formation between human miR-145 (hsa-miR-145; top) and human ADAM17 3′UTR (bottom). (B) Sequence of the miR-145 binding site within the ADAM17 3′UTR of human (Hsa),chimpanzee (Ptr), Rhesus macaque (Mml), mouse (Mmu), and rat (Rno). (C) Relative real-time PCR of miR-145 expression in relation to U48expression in HRCepiC, RCC4, 786-0, A498, and Foehn cells. (D) Western blot analysis for ADAM17 (A17) of total lysates of HRCepiC, RCC4,786-0, A498, and Foehn cells. β-Actin antibody was used as a loading control. The densitometrical analysis of the ADAM17 expression toβ-actin is additionally depicted. (E) Relative real-time PCR of ADAM17 mRNA expression in relation to β-actin expression in HRCepiC, RCC4,786-0, A498, and Foehn cells. (F) Real-time PCR of miR-145 expression relative to U48 expression in RCC tissue compared to normal renaltissue of 15 renal cancer patients (bright gray bars) and real-time PCR of ADAM17 expression in RCC tumor samples compared to normalrenal tissue of the same patients (dark gray bars). The values were evaluated with the Δ-Δ CT method. Correlations were calculated withStudent’s t test, normal tissue against tumor tissue for miR-145 and ADAM17. (G) Plot of fold change of miR-145 related to fold change ofADAM17 for each patient; r, Pearson correlation coefficient; a, regression line. (H) Plot of fold change of miR-145 related to fold change ofc-Myc for each patient. (I) Plot of fold change of miR-145 related to fold change of PAI-1 for each patient. (J) Overview of a tissue section of anormal kidney stained by in situ hybridization for miR-145 (blue) and by immunohistochemistry with an aquaporin-1 (red)–specific antibody.Scale bar represents 300 μm. (K) Enlarged picture of J showing the miR-145–expressing blood vessel. Scale bar represents 300 μm.(L) Enlarged picture of J. Scale bar represents 300 μm. (M) Enlarged picture of I showing miR-145–expressing (blue) and aquaporin-1(red)–expressing tubular cells. Scale bar represents 50 μm. (N) Section of a glomerulus of a normal kidney stained by in situ hybridizationfor miR-145 (blue) and by immunohistochemical analysis for aquaporin-1 (red). Scale bar represents 50 μm.


A498 cells this leads to a decrease of miR-143 expression (Figure 3, B andC). Interestingly, when we transfected the A498 cells with an miR-143inhibitor, it led to a decrease in miR-145 expression (Figure 3D). Surpris-ingly, the inhibition of miR-143 led to a reduction of ADAM17 proteinexpression but showed that miR-143 and miR-145 do not regulateADAM17 synergistically (Figure 3E).

ADAM17 Is Involved in the Effects of miR-145 onProliferation and InvasionTo assess the functional role of miR-145 in renal cancer cells, we

transfected Foehn cells with the miR-145 inhibitor and proliferationwas measured (Figure 4A). Interestingly, miR-145 inhibitor–treatedcells proliferated faster than control cells, whereas transfection with

Figure 1. (continued).


the miR-145 mimic led to a significant reduction in cell prolifera-tion (Figure 4B). A cell cycle analysis was performed to determinethe role of miR-145 in the regulation of the cell cycle. Importantly,the miR-145 mimic led to a 65% increase in cells in sub-G1/G0,whereas the miR-145 inhibitor led to a significant reduction ofcells in sub-G1/G0 (Figure 4C ). The sub-G1/G0 phase representscells that harbor a fragmented nucleus, which is an early event dur-ing apoptosis in which endonucleases start to cleave the genomicDNA [57].We further observed that the amount of S-phase cells decreased after

miR-145 transfection and increased significantly after transfectionwith miR-145 inhibitor. Because the S-phase represents cells duringgenome duplication, it can be used as an indirect measurement of cellproliferation (Figure 4C ). Similar effects on cell cycle and proliferationwere observed in A498 cells (Figure W2, A and B).The role of miR-145 was further analyzed in RCC4 cells, which

expressed the highest miR-145 levels. The transfection of RCC4 cellswith miR-145 mimic and inhibitor was assessed by real-time PCR(Figure W1C ). Interestingly, in contrast to Foehn and A498 cells,

the transfection of miR-145 did not induce apoptosis in RCC4cells (Figure 4D). Although no direct influence on cell death wasobserved, the role of miR-145 in the resistance of renal cancer cellsagainst the chemotherapeutic reagent cisplatin (CDDP) was furtherinvestigated. Transfection of miR-145 mimic significantly increasedthe amount of apoptotic cells after the treatment with CDDP (Fig-ure 4D). In contrast, the transfection with miR-145 inhibitor signifi-cantly reduced the number of cells in sub-G1/G0 CDDP treated anduntreated (Figure 4E).To investigate whether ADAM17 is involved in miR-145–regulated

proliferation, RCC4 cells were transfected with miR-145 inhibitorand additionally with ADAM17 siRNA. The induced proliferation rateafter transfection with miR-145 inhibitor was partially inhibited whenADAM17 was also silenced (Figure 4F). Similarly, increased invasionafter miR-145 inhibition was partly abrogated by the down-regulationof ADAM17 (Figure 4G ). In contrast, miR-145 inhibitor togetherwith ADAM17 siRNA did not change the chemoresistance of RCC4cells against CDDP when compared to the transfection of miR-145inhibitor alone (Figure 4H ). The down-regulation of ADAM17 by

Figure 2. miR-145 binds directly to the 3′UTR of ADAM17 mRNA. (A) Western blot analysis for ADAM17 of total lysates of Foehn cellstransfected with control RNA (ctrl RNA), miR-145 mimic (miR-145), miR-145 inhibitor (miR-145 inh), or specific siRNA against ADAM17(A17-siRNA). β-Actin was used to determine equal loading. The densitometrical analysis of the ADAM17 expression to β-actin is additionallydepicted (n= 4). (B) Western blot analysis for ADAM17 of total lysates of A498 cells transfected with control RNA (ctrl RNA), miR-145mimic(miR-145), miR-145 inhibitor (miR-145 inh), or specific siRNA against ADAM17 (A17-siRNA). β-Actin was used to determine equal loading.The densitometrical analysis of the ADAM17 expression to β-actin is additionally depicted (n = 3). (C) Luciferase assays with reporterconstructs containing either ADAM17 3′UTR with the predicted miR-145 binding site or the mutated binding site. Furthermore, a reporterconstruct carrying the complement sequence of the mature form of miR-145 was transfected as positive control. The Renilla luciferaseactivity for each construct was normalized with firefly luciferase activities. Fold change = (S renilla/S firefly)/(C renilla/C firefly).


miR-145 in RCC4 cells was confirmed by Western blot analysis(Figure 3I). ADAM17 siRNA alone had no effect on proliferation orinvasion in RCC4 cells (Figure W3, A and B).

ADAM17 Regulates miR-145 in a Reciprocal NegativeFeedback LoopBecause it has been demonstrated that various miRNAs, including

miR-145, can be regulated by positive or negative feedback loops,we analyzed whether also ADAM17 is able to regulate miR-145expression [30,58,59].Importantly, the down-regulation of ADAM17 led to a seven-fold in-

duction of miR-145 expression (Figure 5A). One of the most prominentsubstrates of ADAM17 is TNF-α. To analyze whether the increase ofmiR-145 expression was mediated through the reduced cleavage ofTNF-α, we treated the cells with recombinant TNF-α for 1 day, 48 hoursafter siRNA transfection. Importantly, TNF-α reduced the expression ofmiR-145 in the control transfected cells to about 40% (Figure 5A). Incontrast, the seven-fold induction of miR-145 expression after ADAM17down-regulation was abrogated by the addition of recombinant TNF-α(Figure 5A). These results suggest that the reduced cleavage of TNF-αafter ADAM17 down-regulation leads subsequently to the up-regulationof miR-145.To substantiate this hypothesis, we treated the cells with the

metalloprotease inhibitor TAPI-2 or the ADAM17-specific inhibitorTAPI-0, and miR-145 expression was assessed. Importantly, TAPI-0and TAPI-2 increased the miR-145 expression about two-fold(Figure 5B). In contrast, TNF-α and PMA stimulation, which activates

the shedding of ADAM17 substrates, lead to the reduction of miR-145expression. Interestingly, when analyzing both the miR-145 andADAM17 expression after the treatment with TNF-α, the ADAM17expression increases five-fold in contrast to miR-145 expression(Figure 5C). Therefore, our data suggest that the soluble factors cleavedby ADAM17 could be major regulators of miR-145 expression. Theregulation of miR-145 by ADAM17 was further validated in Foehncells (Figure 5D).

miR-145 Is Downregulated by Promoter MethylationBecause it is known from prostate cancer that miR-145 is down-

regulated by methylation, we examined whether methylation couldalso regulate miR-145 expression in renal cancer cells [37]. To test thishypothesis, we treated the cell lines A498, RCC4, and Foehn withAZA for 4 days. AZA leads to the demethylation of the DNA byinhibiting the DNA methyltransferase. AZA treatment resulted in anapproximately 10-fold increase of mature miR-145 in A498 cells anda more than 40-fold increase in RCC4 and Foehn cells (Figure 6A).Because the increase should be reflected by an increase of the primarytranscript, we measured the pri-miR-145, which shows no differencein A498 cells but a three-fold up-regulation in RCC4 and a six-foldup-regulation in Foehn cells (Figure 6B). In contrast, mature miR-143expression, which is transcribed in a cluster with miR-145, showed nochange in these cell lines (Figure 6C).To determine whether the increase of miR-145 after demethylation

regulates also ADAM17 expression, we measured ADAM17 proteinexpression after AZA treatment. In line with the previous results, all

Figure 3. Inhibition of miR-143 reduces ADAM17 expression. (A) Relative real-time PCR of miR-143 expression in relation to U48 expressionin RCC4, Foehn, and A498 cells. (B) Relative real-time PCR of miR-143 expression in relation to U48 expression in Foehn cells that weretransfected with control RNA (ctrl RNA) or miR-145 mimic (miR-145). (C) Relative real-time PCR of miR-143 expression in relation to U48expression in A498 cells that were transfected with control RNA (ctrl RNA) or miR-145 mimic (miR-145). (D) Relative real-time PCR ofmiR-143 and miR-145 expression in relation to U48 expression in A498 cells that were transfected with control LNA (ctrl LNA) ormiR-143 LNA. (E) Western blot analysis for ADAM17 (A17) of total lysates of D. β-Actin antibody was used as a loading control.


tested renal cancer cell lines displayed a reduced ADAM17 expressionafter AZA treatment (Figures 6D and 7).

DiscussionRCC is characterized by its resistance to current standard ther-apies [60]. Despite the rapid progress in the understanding ofthe molecular mechanisms of RCC, new treatment strategies areurgently needed.

In this context, ADAM metalloproteases are a promising family ofproteins that are upregulated in several cancers [61]. miRNAs havebeen shown to be involved in the regulation of members of the ADAMfamily [16–18]. Furthermore, it has been demonstrated that thenon-coding miRNAs have a versatile role for the regulation of genesduring cancer development [62]. In the present study, miR-145 wasidentified as an miRNA that negatively regulates ADAM17. Thebinding was predicted by three different computational algorithms,providing a high probability that miR-145 would also regulate

Figure 4. miR-145 reduces proliferation by induction of apoptosis. (A) Foehn cells were transfected with control RNA (ctrl RNA) or miR-145inhibitor (miR-145 inh). Twenty-four hours after RNA transfection, anchorage-dependent cell growth was measured at the time points 24,48, and 72 hours after RNA transfection using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n = 3). (B) Foehncells were transfected with control RNA (ctrl RNA) or miR-145 mimic (miR-145). Twenty-four hours after RNA transfection, anchorage-dependent cell growth was measured at the time points 24, 48, and 72 hours after siRNA transfection using an MTT assay (n = 3).(C) Foehn cells were transfected with control RNA (ctrl RNA), miR-145 mimic (miR-145), or miR-145 inhibitor (miR-145 inh). Cell cycle analy-sis was performed as described in Materials and Methods section. The percentages of Foehn cells in the sub-G1/G0 (apoptosis), G1 (haploidgenome), S (DNA synthesis), or G2 (diploid genome) phase are depicted (n = 3). (D) RCC4 cells were transfected with control RNA (ctrlRNA) or miR-145 mimic (miR-145). Forty-eight hours after transfection, 10 μg/ml cisplatin (CDDP) was added to the cells for 24 hours. Cellswere harvested andmeasured by cell cycle analysis as described inMaterials andMethods section. The percentage of cells in the sub-G1/G0

(apoptosis), G1 (haploid genome), S (DNA synthesis), or G2 (diploid genome) phase is displayed in a graph (n = 3). (E) RCC4 cells weretransfected with control RNA (ctrl RNA) or miR-145 inhibitor (miR-145 inh). Forty-eight hours after transfection, 10 μg/ml CDDP was addedto the cells for 24 hours. Cells were harvested and measured by cell cycle analysis as described in Materials and Methods section. Thepercentage of cells in the sub-G1/G0 (apoptosis), G1 (haploid genome), S (DNA synthesis), or G2 (diploid genome) phase is displayed (n =3). (F) RCC4 cells were transfected with control RNA (ctrl RNA), miR-145 inhibitor (miR-145 inh), or miR-145 inhibitor with ADAM17 siRNA(miR-145 inh + A17si). Twenty-four hours after RNA transfection, anchorage-dependent cell growth was measured at the time points 24,48, and 72 hours after RNA transfection using anMTT assay. (G) RCC4 cells were transfected with control RNA (ctrl RNA), miR-145 inhibitor(miR-145 inh), or ADAM17 siRNA together with miR-145 inhibitor (miR-145 inh + A17si). Invasion assay was performed using matrigel-coated transwell chambers. (H) RCC4 cells were transfected with control RNA (ctrl RNA), miR-145 inhibitor (miR-145 inh), or ADAM17 siRNAtogether with miR-145 inhibitor (miR-145 inh + A17si). The percentage of cells in the sub-G1/G0 (apoptosis) phase is displayed in a graph.(I) RCC4 cells were transfected with control RNA (ctrl RNA), miR-145 inhibitor (miR-145 inh), or ADAM17 siRNA together with miR-145inhibitor (miR-145 inh + A17si). Cell lysates were investigated by Western blot using antibodies against ADAM17 (A17) and β-actin as aloading control. The densitometrical analysis of the ADAM17 expression to β-actin is additionally depicted. *P < .05, **P < .01, andP < .001 are considered statistically significant. ns, not significant.


ADAM17 directly under physiological conditions. The binding hasbeen additionally suggested by Gregersen et al. [41]. The regulationof ADAM17 by miR-145 was shown in different renal cancer celllines by the transfection of miR-145 mimic as well as by inhibitingmiR-145 expression. The overexpression of miR-145 led to massivereduction in ADAM17 protein expression, whereas the inhibition ofmiR-145 increased the amount of ADAM17, demonstrating the im-portance of miR-145 in the regulation of ADAM17. By performingmRNA reporter assay, we could show that miR-145 can directly bindto the predicted 3′UTR of ADAM17 mRNA.The idea that miR-145 has also a role in the regulation of ADAM17

was supported by the finding that cancer cell lines have reducedmiR-145expression and increased ADAM17 expression when compared to renaltubular control cell line. Importantly, miR-145 is downregulated inmany cancers, whereas different studies demonstrated that ADAM17

was upregulated in the same cancer types [3–11,25–28]. In this study,we could show that, in most renal cancer patients, miR-145 is down-regulated, whereas ADAM17 is upregulated. Moreover, the analysis ofRCC patients revealed a significantly negative correlation of ADAM17and miR-145, indicating that both genes are tightly regulated. Interest-ingly, the patient that was not included in the correlation had the worstclinical outcome with the highest number of metastases. This patientexpressed the lowest relative amount of ADAM17 and miR-145, whichcould indicate that at this late stage ADAM17 might be downregulatedby factors independent of miR-145. In agreement with our results,the down-regulation of miR-145 has been considered as an early eventin other cancers, which has an influence on several tumor-promotingpathways [31,32].miR-145 has been shown to be expressed in vascular SMCs, playing

an essential role for smooth muscle development [22–24]. Similarly,

Figure 4. (continued).


we could detect strong miR-145 expression in vascular SMCs of thekidney. Additionally, we observed miR-145 expression in glomeruliand renal tubules, where the predominantly smaller tubules were posi-tive for miR-145. In addition to the glomeruli, the Bowman’s capsuleand adjacent tubules expressed miR-145. These data suggest a role formiR-145 in normal adult glomeruli and renal tubules.Our findings that miR-145 reduces proliferation and induces

apoptotic cell death in renal cancer cells are in agreement with theresults in other cancer types [36,58,63]. It was further demonstratedthat ADAM17 is partially involved in the effects of miR-145 onproliferation and invasion, suggesting a role of additional target genes.Particularly, the regulation of the p53 repressor MDM2 by miR-145could play a major role for the changes in cell cycle, chemoresistance,and proliferation [58]. Additionally, it was shown that miR-145 targetsP70S6K1, which is important for the activation of the phosphatidyl-inositide 3-kinases (PI3K)/Akt pathway, resulting in reduced tumorgrowth [36].We also demonstrated that not only miR-145 negatively regulates

ADAM17 but ADAM17 also downregulates the miR-145 reciprocalnegative feedback loop. Positive and negative feedback loops have been

described for many miRNAs, including miR-145 [30,58,59]. Similarlyto our work, Xu and colleagues observed a reciprocal negative feed-back loop of miR-145 and Oct4, resulting either in pluripotent cellswith low miR-145 expression or differentiated cells with high levelsof miR-145 [35].Reciprocal negative and positive feedback loops are often asso-

ciated with a cell type switch that leads to a long-lasting cellularresponse [64,65].Importantly, we showed that this regulation is transmitted by cleaved

substrates of ADAM17, like TNF-α. TNF-α levels are often increasedin the tumor microenvironment by tumor cells and by stroma cells,leading to a more tumorigenic tumor and increased metastasis. Itwould be interesting to investigate whether inhibition of the ADAM17cleavage by specific inhibitors could reduce the cleaved substrates inthe microenvironment and increase the miR-145 expression in tumorcells in vivo, thus resulting in better treatment options. Similar toour results, up-regulation of miR-145 in many cancer types inducesantitumor properties like sensitivity toward chemotherapeutic treat-ment [26,29,30]. Moreover, we demonstrated that miR-145 is down-regulated by methylation, which is in agreement with studies in other

Figure 5. ADAM17 regulates miR-145 expression by cleavage of TNF-α. (A) Real-time PCR of miR-145 relative to U48 expression in RCC4cells after transfection with either control RNA (ctrl RNA) or specific ADAM17 siRNA (A17-siRNA). Forty-eight hours after transfection, thecells were treated with control vehicle (bright bars) or with 10 nM TNF-α (dark bars). The Western blot analysis for ADAM17 (A17) of totallysates to control the efficient down-regulation is additionally depicted. β-Actin antibody was used as a loading control. (B) Real-time PCR ofmiR-145 expression relative to U48 expression in RCC4 cells after treatment for 24 hours with either control vehicle (ctrl), PMA (100 ng/ml),TNF-α (10 nM), or the metalloprotease inhibitors TAPI-0 and TAPI-2 (both 10 μM). *P < .05, **P < .01, and ***P < .001 are consideredstatistically significant. (C) Real-time PCR of miR-145 expression relative to U48 expression (gray bars) and ADAM17 expression to β-actinexpression (black bars) in RCC4 cells after treatment for 24 hours with either control vehicle (ctrl) or TNF-α (10 nM). (D) Real-time PCR ofmiR-145 expression relative to U48 expression in Foehn cells after transfection with either control RNA (ctrl RNA, bright bars) or specificADAM17 siRNA (A17-siRNA, dark bars). TheWestern blot analysis for ADAM17 (A17) of total lysates to control the efficient down-regulationis additionally depicted. β-Actin antibody was used as a loading control.


cancer types [37–39]. The strong induction of miR-145 after theaddition of the demethylation agent in three cell lines indicates a directmethylation of miR-145 and not a secondary effect through a sec-ondary regulator. Interestingly, we observed an increase of maturemiR-145 that was about 10 times higher than the amount of the

primary transcript. It could be that the treatment with AZA alsoincreases the processing of primary miR-145 to the mature form. Incontrast, no strong differences in mature miR-143, which is transcribedas a cluster together with miR-145, were observed, and this might bedue to differences in processing.

Figure 6. miR-145 is downregulated by methylation in renal cancer cells. (A) Real-time PCR of miR-145 expression relative to U48 ex-pression in A498, RCC4, and Foehn cells after treatment with either control vehicle (ctrl, bright bars) or AZA (10 μM, dark bars) for72 hours. (B) Real-time PCR of pri-miR-145 expression relative to β-actin expression in A498, RCC4, and Foehn cells after treatmentwith either control vehicle (ctrl, bright bars) or AZA (10 μM, dark bars) for 72 hours. (C) Real-time PCR of miR-143 expression relativeto U48 expression in A498, RCC4, and Foehn cells after treatment with either control vehicle (ctrl, bright bars) or AZA (10 μM, dark bars)for 72 hours. (D) A498, RCC4, and Foehn cells after treatment with either control vehicle (−) or AZA (10 μM, +) for 72 hours. Cell lysateswere investigated by Western blot analysis with antibodies against ADAM17 and β-actin as a loading control.

Figure 7. Reciprocal negative feedback loop of miR-145 and ADAM17 is transmitted through TNF-α. (A) Endogenous expression ofmiR-145 in normal kidney cells leads to the down-regulation of ADAM17 expression. As a consequence, less substrates are releasedby ADAM17, leading to an even higher expression of miR-145. As a result, ADAM17 is weakly expressed, whereas the miR-145 expres-sion is high. (B) miR-145 expression is suppressed in renal cancer cells by an initial event, like promoter methylation. This leads to anup-regulation of ADAM17 expression, resulting in an increase of ADAM17-mediated cleavage of substrates like TNF-α. The releasedsubstrates then further suppress the expression of miR-145, leading to a reciprocal negative feedback loop. As a result, ADAM17expression is elevated in renal cancer patients, whereas miR-145 is downregulated.


In the future, it would be important to analyze the methyla statusof renal cancer patients. Given that, demethylation agents could bean important treatment option to increase miR-145 expression inrenal cancer cells and thus sensitize renal cancer cells for therapy.In summary, the data of the present work demonstrated that

miR-145 is a tumor-suppressive miRNA in RCC that regulates themetalloprotease ADAM17. It could be a novel treatment strategy toincrease the miR-145 expression by demethylation or by inhibitingthe ADAM17 cleavage in renal cancer patients.

AcknowledgmentsWe thank Nicole Kämpfer-Kolb for her excellent technical assis-tance. We also thank Dario Alejandro Gutierrez for his great helpand manuscript corrections.

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Figure W1. (A) Real-time PCR of miR-145 relative to U48 expression in Foehn cells after the transfection with control RNA (ctrl RNA),miR-145 mimic (miR-145), or miR-145 inhibitor (miR-145 inh). (B) Real-time PCR of miR-145 relative to U48 expression in A498 cells afterthe transfection with control RNA (ctrl RNA), miR-145 mimic (miR-145), or miR-145 inhibitor (miR-145 inh). (C) Real-time PCR of miR-145expression relative to U48 expression in RCC4 cells after transfection with either control RNA (ctrl RNA), miR-145 mimic (miR-145), ormiR-145 inhibitor (miR-145 inh).

Figure W2. (A) A498 cells were transfected with control RNA (ctrl RNA) or miR-145 mimic (miR-145). Twenty-four hours after RNA trans-fection, anchorage-dependent cell growth was measured at the time points 24, 48, and 72 hours after siRNA transfection using an MTTassay (n = 3). (B) A498 cells were transfected with control RNA (ctrl RNA) or miR-145 mimic (miR-145). Cell cycle analysis was performedas described in Materials and Methods section. The percentages of Foehn cells in the sub-G1/G0 (apoptosis), G1 (haploid genome),S (DNA synthesis), or G2 (diploid genome) phase are depicted (n = 3). **P < .01 and ***P < .001 are considered statistically significant.ns, not significant.

Figure W3. (A) RCC4 cells were transfected with control RNA (ctrl RNA) or ADAM17 siRNA (A17-siRNA). Twenty-four hours after RNAtransfection, anchorage-dependent cell growth was measured at the time points 24, 48, and 72 hours after RNA transfection using anMTT assay. (B) RCC4 cells were transfected with control RNA (ctrl RNA) or ADAM17 siRNA (A17-siRNA). Invasion assay was performedusing matrigel-coated transwell chambers.

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MicroRNA-145 Targets the Metalloprotease ADAM17 and Is Suppressed in Renal Cell Carcinoma Patients