MicroRNAs are emerging as important regulators of cancer-related processes. The miR-21 microRNA is overexpressed in a wide variety of cancers and has been causally linked to cellular proliferation, apoptosis, and migration. Inhibition of mir-21 in MCF-7 breast cancer cells causes reduced cell growth. Using array expression analysis of MCF-7 cells depleted of miR-21, we have identified mRNA targets of mir-21 and have shown a link between miR-21 and the p53 tumor suppressor protein. We furthermore found that the tumor suppressor protein Programmed Cell Death 4 (PDCD4) is regulated by miR-21 and demonstrated that PDCD4 is a functionally important target for miR-21 in breast cancer cells. MicroRNAs are emerging as important regulators of cancer-related processes. The miR-21 microRNA is overexpressed in a wide variety of cancers and has been causally linked to cellular proliferation, apoptosis, and migration. Inhibition of mir-21 in MCF-7 breast cancer cells causes reduced cell growth. Using array expression analysis of MCF-7 cells depleted of miR-21, we have identified mRNA targets of mir-21 and have shown a link between miR-21 and the p53 tumor suppressor protein. We furthermore found that the tumor suppressor protein Programmed Cell Death 4 (PDCD4) is regulated by miR-21 and demonstrated that PDCD4 is a functionally important target for miR-21 in breast cancer cells. Since their discovery (1Lee R.C. Feinbaum R.L. Ambros V. Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (9449) Google Scholar, 2Lee R.C. Ambros V. Science. 2001; 294: 862-864Crossref PubMed Scopus (2291) Google Scholar, 3Lau N.C. Lim L.P. Weinstein E.G. Bartel D.P. Science. 2001; 294: 858-862Crossref PubMed Scopus (2652) Google Scholar, 4Lagos-Quintana M. Rauhut R. Lendeckel W. Tuschl T. 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In animals, miRNAs bind to partly complementary sequence motifs present predominantly within the 3′-untranslated regions (UTRs) and mediate translational repression, sometimes involving degradation of the target mRNA (7Bagga S. Bracht J. Hunter S. Massirer K. Holtz J. Eachus R. Pasquinelli A.E. Cell. 2005; 122: 553-563Abstract Full Text Full Text PDF PubMed Scopus (1095) Google Scholar, 8Jackson R.J. Standart N. Sci. STKE. 2007; 2007: re1Crossref PubMed Scopus (364) Google Scholar, 9Wu L. Fan J. Belasco J.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4034-4039Crossref PubMed Scopus (902) Google Scholar). miRNAs have been found implicated in a multitude of cellular processes including proliferation, differentiation, migration, and apoptosis (10Kloosterman W.P. Plasterk R.H. Dev. Cell. 2006; 11: 441-450Abstract Full Text Full Text PDF PubMed Scopus (1772) Google Scholar, 11Meng F. Henson R. Wehbe-Janek H. Ghoshal K. Jacob S.T. Patel T. 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This is exemplified by the analysis of let-7, which in Caenorhabditis elegans targets let-60/RAS and in humans is lost in some lung cancers, leading to overexpression of N-RAS (23Johnson S.M. Grosshans H. Shingara J. Byrom M. Jarvis R. Cheng A. Labourier E. Reinert K.L. Brown D. Slack F.J. Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar). Third, forward genetic studies have demonstrated a role for the miR-17-92 cluster in lymphoma development in mice predisposed to cancer (24He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. Nature. 2005; 435: 828-833Crossref PubMed Scopus (3120) Google Scholar), and screenings of miRNA expression libraries in cell culture models of cancer have identified miR-372/373 and miR-221/222 as cancer-promoting miRNAs via repression of the tumor suppressor proteins LATS2 and p27, respectively (25le Sage C. Nagel R. Egan D.A. Schrier M. Mesman E. Mangiola A. Anile C. Maira G. Mercatelli N. Ciafre S.A. Farace M.G. Agami R. EMBO J. 2007; 26: 3699-3708Crossref PubMed Scopus (692) Google Scholar, 26Voorhoeve P.M. le Sage C. Schrier M. Gillis A.J. Stoop H. Nagel R. Liu Y.P. van Duijse J. Drost J. Griekspoor A. Zlotorynski E. Yabuta N. De Vita G. Nojima H. Looijenga L.H. Agami R. Cell. 2006; 124: 1169-1181Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar).MicroRNA profiling studies of human tumors have shown cancer type-specific deregulation of miRNA expression and have identified a number of miRNAs with putative tumor suppressor or oncogenic functions (13Lu J. Getz G. Miska E.A. Alvarez-Saavedra E. Lamb J. Peck D. Sweet-Cordero A. Ebert B.L. Mak R.H. Ferrando A.A. Downing J.R. Jacks T. Horvitz H.R. Golub T.R. Nature. 2005; 435: 834-838Crossref PubMed Scopus (8125) Google Scholar, 15Calin G.A. Croce C.M. Nat. Rev. Cancer. 2006; 6: 857-866Crossref PubMed Scopus (6521) Google Scholar). 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The widespread occurrence of miR-21 overexpression in cancer and the relatively few experimentally defined targets prompted us to perform expression array analyses of breast cancer cells transfected with miR-21 inhibitors to identify and functionally validate additional targets of miR-21.EXPERIMENTAL PROCEDURESAntibodies and Western Blot Analysis—MCF-7 cells were seeded in 6-well plates and transfected the following 2 days. The cells were harvested 5 days after the first transfection, washed once in phosphate-buffered saline, and lysed in radioimmune precipitation buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-HCl, pH 8, 2 mm EDTA) containing 1 mm dithiothreitol, 1 mm Pefabloc (Roche Applied Science), 1× Complete Mini protease inhibitor mixture (Roche Applied Science), 1 mm NaVO3, 10 mm NaF, 10 mm pyrophosphate, and 50 mm β-glycerophosphate. 15 μg of protein/lane was separated on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The PDCD4 antibody was kindly provided by Dr. Iwata Ozaki, Japan. The p53 and CDK6 antibodies were purchased from Santa Cruz Biotechnology. The cofilin 2 antibody was purchased from Cell Signaling and the vinculin antibody from Sigma-Aldrich.miRNA Precursors, Anti-miRNA Oligonucleotides, and siRNAs—The locked nucleic acid (LNA)-modified oligonucleotide inhibitors used for miRNA knock down were purchased from Exiqon. The miRNA precursor hairpins were purchased from Ambion. The PDCD4 SMARTpool siRNA was purchased from Dharmacon.Cell Culture—HEK293 and MCF-7 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Biochrom), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) and incubated at 37 °C in 5% CO2. MCF-7 cells expressing an ecotropic receptor were transduced with pRetroSuper-shp53 (34Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3945) Google Scholar) or the empty pRetroSuper virus to obtain the MCF-7 shp53 and MCF-7 EV cell lines, respectively. For proliferation assays, MCF-7 cells were transferred to 1.0% fetal bovine serum prior to transfections with miRNA inhibitors.Vector Construction and Reporter Assays—A multiple cloning site was inserted into the pGL3 control vector (Invitrogen) at the XbaI site 3′ of the luciferase gene (hereafter pGL3). Six different fragments were PCR-amplified from human genomic DNA and cloned into pGL3. The primer sequences used for PCR amplification were as follows (restriction sites are underlined). SOCS5 FW, 5′-gggagatctgactgctaatgtatgtttct-3′; SOCS5 RV, 5′-gggctcgagtactgatctattacaaattc-3′. IL6R FW, 5′-gggagatctctgcagtaagggtgattctg-3′; IL6R RV, 5′-gggctcgagccagcaggataagctgtcgta-3′. BTG2 FW, 5′-gggagatctgtattgccttcccagacctg-3′; BTG2 RV, 5′-gggctcgagaaggtgtacatttgtccata-3′. CDK6 FW, 5′-gggagatcttcaagttgttctacatttgc-3′; CDK6 RV, 5′-gggctcgagcaggcactggtctctgcctg-3′. BMPRII FW, 5′-gggagatcttatgtaagctggaatcatcc-3′; BMPRII RV, 5′-gggctcgagtagtggcttaatgtcagctt-3′. PDCD4 FW, 5′-gggtctagagacattttataaacctacat-3′; PDCD4 RV, 5′-aatcaatactgcttcacatg-3′.The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce one or two point mutations into the seed region of pGL3-PDCD4, giving pGL3-PDCD4MUT1 and pGL3-PDCD4MUT2, respectively. Mutagenesis primers used were as follows. MUT1 FW, 5′-gtggaatattctaataaggtaccttttgtaagtgccatg-3′; MUT1 RV, 5′-catggcacttacaaaaggtaccttattagaatattccac-3′. MUT2 FW, 5′-gtggaatattctaataacgtaccttttgtaagtgccatg-3′; MUT2 RV, 5′-catggcacttacaaaaggtacgttattagaatattccac-3′. The pmiR-21-luc reporter vector was constructed by inserting an oligo complementary to the mature miR-21 sequence into the pMIR-REPORT luciferase vector (Ambion).HEK293 and MCF-7 cells were seeded in 96-well plates and transfected with 50 nm miRNA precursor or LNA, 100-150 ng of luciferase vector (pGL3 constructs), and 25 ng of Renilla vector (pRL-TK) using Lipofectamine 2000 (Invitrogen). 24 h after transfection, cells were harvested and luciferase activity was measured using the Dual-Glo luciferase assay (Promega).Quantitative PCR Analysis—Total RNA from transfected cells was isolated with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Quantitative reverse transcription PCR was performed using the TaqMan reverse transcription kit (Applied Biosystems) and Sybr Green 2× quantitative PCR master mix (Applied Biosystems). PCR primers used were as follows: FAM3C FW, 5′-gctgggaggccggagcata-3′ and RV, 5′-tgcagcacctgctaccctcatg-3′. HIPK3 FW, 5′-ctctacccaggagccttggagtat-3′ and RV, 5′-tgttctcctggcaaaccttgagtct-3′; PRRG4 FW, 5′-atgcgggagaagaagtgtttacatca-3′ and RV, 5′-gggagtgaagagctccagatcaaatc-3′. RP2 FW, 5′-tcagcgcgagaaggttgatcc-3′ and RV, 5′-caggtaagcgacctactgtttcatcc-3′. SGK3 FW, 5′-gttctggtttcagtgggaagaagtga-3′ and RV, 5′-agggccatagcaggaaactgtttt-3′. GLCCI1 FW, 5′-cggaggagcagctcacctgag-3′ and RV, 5′-cgtggccactgtcctgtgaggta-3′. SLC16A10 FW, 5′-tggtctttaagacagcatgggtaggt-3′ and RV, 5′-tgaagacgctgactattgggcag-3′. BMPR-II FW, 5′-atctgtgagcccaacagtcaatcca-3′ and RV, 5′-gaccaatttttggcacacgccta-3′. BTG2 FW, 5′-cgtgagcgagcagaggcttaag-3′ and RV, 5′-tggacggcttttcgggaa-3′. CDK6 FW, 5′-ctgcccaaccaattgagaagtttg-3′ and RV, 5′-cagggcactgtaggcagatattcttt-3′. IL6R FW, 5′-gggctctgaaggaaggcaagaca-3′ and RV, 5′-cggtggggagatgagaggaaca-3′. SOCS5 FW, 5′-atctgtaacctcccactgcatcagaa-3′ and RV, 5′-gatggtccccaaaccaataaatgg-3′. PDCD4 FW, 5′-tatgatgtggaggaggtggatgtga-3′ and RV, 5′-cctttcatccaaaggcaaaactacac-3′. ACTA2 FW, 5′-agcacatggaaaagatctggcacc-3′ and RV, 5′-ttttctcccggttggccttg-3′. APAF-1 FW, 5′-tctgatgcttcgcaaacaccca-3′ and RV, 5′-ctttcaacacccaagagtcccaaa-3′. FAS FW, 5′-cctccaggtgaaaggaaagctagg-3′ and RV, 5′-ttcttggcagggcacgca-3′. SESN1 FW, 5′-actacattggaataatggctgcgg-3′ and RV, 5′-ccccaccaacatgaaggaaatcat-3′. CDKN1A FW, 5′-ggcagaccagcatgacagatttc-3′ and RV, 5′-cggattagggcttcctcttgg-3′.Growth Curves and Viability Assays—MCF-7 cells were seeded in 24-well plates and transfected the following day with 50 nm LNA or siRNA using Lipofectamine 2000. Cells were fixed at indicated time points in 4% paraformaldehyde, stained in a 0.1% crystal violet solution, and resuspended in 10% acetic acid. Sample absorbance was measured at 620 nm. For cell viability assays, MCF-7 cells were seeded in 24-well plates and transfected the following day with 50 nm LNA or siRNA using Lipofectamine 2000. After 5-6 days of growth, cells were incubated for 4 h at 37 °C with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich). After incubation, supernatants were discarded and formazan crystals were dissolved in a 10% formic acid, 90% isopropanol solution. Sample absorbance was measured at 570 nm with a reference wavelength of 690 nm.Affymetrix Microarray—MCF-7 wells were seeded in 10-cm plates in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and triplicate independent transfections performed the following day with either 50 nm LNA-miR-21 or scrambled control LNA using Lipofectamine 2000. Total RNA was harvested 24 h post-transfection using TRIzol reagent. Affymetrix microarray analysis (HG-U133 Plus 2.0 human) was performed at the Microarray Center, Rigshospitalet, Copenhagen University Hospital. Briefly, 2 μg of total RNA was used to synthesize double-stranded cDNA using Superscript® Choice System (Invitrogen) with an oligo(dT) primer containing a T7 RNA polymerase promoter. The cDNA was subsequently used as template for an “in vitro” transcription reaction generating biotin-labeled antisense cRNA (BioArrayTM High Yield RNA Transcript Labeling kit; Enzo Diagnostics, Farmingdale, NY). After fragmentation at 94 °C for 35 min in 40 mm Tris, 30 mm MgOAc, 10 mm KOAc, samples were hybridized for 16 h to Affymetrix HG-U133 2.0 human arrays (Affymetrix, Santa Clara, CA). The arrays were washed and stained with phycoerythrin-conjugated streptavidin (SAPE), and the arrays were scanned in the Affymetrix GeneArray® 2500 scanner, exactly as described in the Affymetrix GeneChip® protocol.Bioinformatic Analyses—The expression data were processed using the “affy” package in BioConductor. Probe set intensities were summarized using the Robust Multichip Average method and then transformed to generalized log values (approaches the natural logarithm for high values) with the variance stable VSN method (35Huber W. von Heydebreck A. Sultmann H. Poustka A. Vingron M. Bioinformatics. 2002; 18: S96-S104Crossref PubMed Scopus (1598) Google Scholar). In a final step, the six arrays were qspline-normalized. Differentially expressed genes were selected by a t test (p < 0.05), and two sets of probe sets were defined, the up and down-regulated, by additionally requiring reasonable average expression intensities (>2.87, 1st quartile) and high -fold changes (>± 0.168, mean ± 2 × S.D.) (supplemental Table S1, a and b). A third set of probe sets that do not change from control to experiment was defined by selecting probe sets with stable expression intensities (p > 0.95 and expression intensity S.D. < 0.06) and reasonable expression (>2.87) (supplemental Table S1c). The up, down, and no-change sets comprised 339, 258, and 195 probe sets, respectively.The probe sets were subsequently mapped to Ensembl transcripts (version 45) using the mappings provided at BioMart. Probe sets that mapped to two different Ensembl genes were discarded. Transcripts with 3′-UTR sequences shorter than 50 nucleotides and copies of transcript isoforms of the same gene with matching 3′-UTR sequences were discarded. The up, down, and no-change sets comprised 402, 335, and 262 transcripts, corresponding to 272, 215, and 164 genes. For the analysis of seed site enrichment, miRNA sequences from miRBase version 9.2 were used (36Griffiths-Jones S. Methods Mol. Biol. 2006; 342: 129-138Crossref PubMed Scopus (523) Google Scholar). The 3′-UTRs of the transcripts were scanned for matching 6-, 7-1A, 7-, and 8-mer (perfect 8-nucleotide match) miRNA seed sites (37Grimson A. Farh K.K. Johnston W.K. Garrett-Engele P. Lim L.P. Bartel D.P. Mol. Cell. 2007; 27: 91-105Abstract Full Text Full Text PDF PubMed Scopus (2994) Google Scholar). In the definition used, seed sites are contained in each other, that is, a given 6-mer site always also corresponds to a 7 mer and 8 mer. Note that this definition is different from the seed sites reported at TargetScan. The difference in the fraction of transcripts having seed sites in the up, down, and no-change sets was evaluated using one-sided p values from Fisher's exact test. When comparing all miRBase miRNAs, the -fold enrichment for a given miRNA is calculated by dividing the fraction of transcripts with a 7-mer seed site in the up set with the same fraction in the down (or no-change) set.In an unbiased word analysis, all words of length 7 were investigated for over-representation in the up versus the down sets. The words were ranked by p values from Fishers exact test.RESULTSSuppression of MCF-7 Cell Growth by Inhibition of miR-21—Several studies have demonstrated that miR-21 is an oncogenic miRNA with anti-apoptotic potential. Inhibition of miR-21 leads to growth suppression and apoptosis in glioblastoma and breast cancer cell lines, and loss of miR-21 can inhibit MCF-7 cell-derived tumor growth in vivo (30Chan J.A. Krichevsky A.M. Kosik K.S. Cancer Res. 2005; 65: 6029-6033Crossref PubMed Scopus (2201) Google Scholar, 31Si M.L. Zhu S. Wu H. Lu Z. Wu F. Mo Y.Y. Oncogene. 2007; 26: 2799-2803Crossref PubMed Scopus (1350) Google Scholar, 33Meng F. Henson R. Lang M. Wehbe H. Maheshwari S. Mendell J.T. Jiang J. Schmittgen T.D. Patel T. Gastroenterology. 2006; 130: 2113-2129Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar). MCF-7 cells express substantial amounts of miR-21 (supplemental Fig. S1), and consistent with previous findings, we observed a dose-dependent suppression of MCF-7 cell growth upon inhibition of miR-21 with an LNA-derived oligonucleotide inhibitor (Fig. 1A). Co-transfection of the LNA inhibitor with a luciferase reporter containing perfect complementarity to the mature miR-21 sequence (pmiR-21-luc) results in marked de-repression of luciferase activity, demonstrating a highly effective inhibition of endogenous miR-21 mediated by the LNA inhibitor (supplemental Fig. S2). The underlying mechanism of the role of miR-21 in tumorigenesis remains unclear, as only few targets for this miRNA have been experimentally verified (11Meng F. Henson R. Wehbe-Janek H. Ghoshal K. Jacob S.T. Patel T. Gastroenterology. 2007; 133: 647-658Abstract Full Text Full Text PDF PubMed Scopus (2317) Google Scholar, 32Zhu S. Si M.L. Wu H. Mo Y.Y. J. Biol. Chem. 2007; 282: 14328-14336Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar). Meng et al. (11Meng F. Henson R. Wehbe-Janek H. Ghoshal K. Jacob S.T. Patel T. Gastroenterology. 2007; 133: 647-658Abstract Full Text Full Text PDF PubMed Scopus (2317) Google Scholar) have recently shown that the tumor suppressor PTEN is a direct functional target of miR-21 in human hepatocellular cancer cell lines. Given the importance of PTEN in regulating the PI3K/AKT pathway and the frequency of PTEN mutations or silencing in a variety of cancers (38Chow L.M. Baker S.J. Cancer Lett. 2006; 241: 184-196Crossref PubMed Scopus (236) Google Scholar), this constitutes an appealing explanation for the overexpression of miR-21 observed in many cancer types (27Volinia S. Calin G.A. Liu C.G. Ambs S. Cimmino A. Petrocca F. Visone R. Iorio M. Roldo C. Ferracin M. Prueitt R.L. Yanaihara N. Lanza G. Scarpa A. Vecchione A. Negrini M. Harris C.C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2257-2261Crossref PubMed Scopus (4895) Google Scholar, 28Roldo C. Missiaglia E. Hagan J.P. Falconi M. Capelli P. Bersani S. Calin G.A. Volinia S. Liu C.G. Scarpa A. Croce C.M. J. Clin. Oncol. 2006; 24: 4677-4684Crossref PubMed Scopus (664) Google Scholar). To investigate the role of the PTEN-miR-21 interaction in breast cancer cells, we transfected MCF-7 cells with a miR-21 precursor, a miR-21 inhibitor, and appropriate controls. Interestingly, these treatments caused only subtle changes in PTEN protein levels (supplemental Fig. S3), suggesting that cell- and tissue type-specific differences may result in different functional miR-21 targets.Identification and Validation of miR-21 Targets by Microarray Analysis—To identify targets of miR-21 that can explain the proliferation defect observed upon miR-21 inhibition in breast cancers cells, we performed microarray expression analyses using total RNA harvested from MCF-7 cells 24 h after transfection with either LNA-miR-21 or a scrambled control LNA not affecting cellular proliferation (Fig. 1B). We reasoned that cellular mRNAs subjected to increased degradation due to binding of endogenous miR-21 would be up-regulated upon miR-21 inhibition and that specific inhibition of the endogenous miR-21 may cause fewer off-target effects than transfection with exogenous miRNA. Following normalization and statistical analysis we found 737 transcripts with significantly different expression between the LNA-miR-21-transfected cells and the controls, of which 402 (55%) were up-regulated and 335 (45%) down-regulated upon miR-21 inhibition (Fig. 2A and supplemental Table S1). To verify the array analysis, 18 up-regulated mRNAs were validated by quantitative reverse transcription PCR analysis of RNA from an independent experiment. Ten of the genes chosen for validation contained at least one miR-21 7-mer seed match, two contained a 6-mer, and six did not contain miR-21 seed matches in their 3′-UTR. All 18 genes showed, to varying degrees, increased mRNA expression levels upon miR-21 inhibition (Fig. 2B). It is unclear whether the mRNAs without matches to the miR-21 seed sequence are direct targets or whether their regulation is a result of secondary effects.FIGURE 2Identification of miR-21 targets.A, cluster heat map of Affymetrix microarray analyses of six independent biological samples showing the 402 transcripts significantly up-regulated by miR-21 inhibition in red and the 335 significantly down-regulated transcripts in blue. B, quantitative reverse transcription PCR validation of 18 up-regulated transcripts from an independent transfection experiment. CDKN1A, FAS, FAM3C, HIPK3, PRRG4, and ACTA2 do not contain matches to the miR-21 seed region, BTG2 and SESN1 contain a 6-mer seed match, and the remaining mRNAs harbor at least one 7-mer seed match. For each transcript the values are normalized to the LNA-scrambled control samples, and error bars represent ± S.D. of three replicates. C, the relative fraction of transcripts among the up-, down-, or non-regulated transcripts containing the indicated type of seed match. ***, one-tailed p values from Fisher's exact test are: 6-mer up versus no-change, p < 1 × 10-13; 7-mer-1A up versus no-change, p < 1.5 × 10-12; 7-mer up versus no change, p < 6.1 × 10-16; 8-mer up versus down, p < 9.6 × 10-6. D, frequency distribution of 7-mer seed matches for all miRNAs in miRBase showing a marked over-representation of the miR-21 seed match in the up-versus down-regulated genes. miR-590 and miR-21 have identical seed sequences.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To get a qualitative assessment of the data, we analyzed for the presence of different types of miR-21 seed matches among the genes regulated by miR-21 inhibition. Notably, we found very significant over-representations of all miR-21 seed match categories among the genes up-regulated by miR-21 inhibition relative to gene sets that exhibited no change or down-regulated expression, strongly suggesting that inhibition of endogenous miRNAs can be used to identify bona fide targets (Fig. 2C). In effect, the motif complementary to a 7-mer miR-21 seed sequence (or miR-590, holding the exact same seed sequence) is by far the most prevalent motif when analyzing the 3′-UTRs of the up-regulated transcripts against all seed sequences present in miRBase (36Griffiths-Jones S. Methods Mol. Biol. 2006; 342: 129-138Crossref PubMed Scopus (523) Google Scholar) (Fig. 2D). In addition, an unbiased analysis for the frequency of all possible 7-mer sequence motifs, regardless of whether these match known mi-RNAs, shows that the miR-21 complementary motif is very highly enriched (p < 4 × 10-12) and represents the most frequently occurring sequence motif in the 3′-UTRs of the up-regulated versus the down-regulated transcripts. Hence, the
