MicroRNA-21 Targets the Tumor Suppressor Gene Tropomyosin 1 (TPM1)

MicroRNAs are small noncoding RNA molecules that control expression of target genes. Our previous studies show that mir-21 is overexpressed in tumor tissues compared with the matched normal tissues. Moreover, suppression of mir-21 by antisense oligonucleotides inhibits tumor cell growth both in vitro and in vivo. However, it remains largely unclear as to how mir-21 affects tumor growth, because our understanding of mir-21 targets is limited. In this study, we performed two-dimensional differentiation in-gel electrophoresis of tumors treated with anti-mir-21 and identified the tumor suppressor tropomyosin 1 (TPM1) as a potential mir-21 target. In agreement with this, there is a putative mir-21 binding site at the 3′-untranslated region (3′-UTR) of TPM1 variants V1 and V5. Thus, we cloned the 3′-UTR of TPM1 into a luciferase reporter and found that although mir-21 down-regulated the luciferase activity, anti-mir-21 up-regulated it. Moreover, deletion of the mir-21 binding site abolished the effect of mir-21 on the luciferase activity, suggesting that this mir-21 binding site is critical. Western blot with the cloned TPM1-V1 plus the 3′-UTR indicated that TPM1 protein level was also regulated by mir-21, whereas real-time quantitative reverse transcription-PCR revealed no difference at the mRNA level, suggesting translational regulation. Finally, overexpression of TPM1 in breast cancer MCF-7 cells suppressed anchorage-independent growth. Thus, down-regulation of TPM1 by mir-21 may explain, at least in part, why suppression of mir-21 can inhibit tumor growth, further supporting the notion that mir-21 functions as an oncogene. MicroRNAs are small noncoding RNA molecules that control expression of target genes. Our previous studies show that mir-21 is overexpressed in tumor tissues compared with the matched normal tissues. Moreover, suppression of mir-21 by antisense oligonucleotides inhibits tumor cell growth both in vitro and in vivo. However, it remains largely unclear as to how mir-21 affects tumor growth, because our understanding of mir-21 targets is limited. In this study, we performed two-dimensional differentiation in-gel electrophoresis of tumors treated with anti-mir-21 and identified the tumor suppressor tropomyosin 1 (TPM1) as a potential mir-21 target. In agreement with this, there is a putative mir-21 binding site at the 3′-untranslated region (3′-UTR) of TPM1 variants V1 and V5. Thus, we cloned the 3′-UTR of TPM1 into a luciferase reporter and found that although mir-21 down-regulated the luciferase activity, anti-mir-21 up-regulated it. Moreover, deletion of the mir-21 binding site abolished the effect of mir-21 on the luciferase activity, suggesting that this mir-21 binding site is critical. Western blot with the cloned TPM1-V1 plus the 3′-UTR indicated that TPM1 protein level was also regulated by mir-21, whereas real-time quantitative reverse transcription-PCR revealed no difference at the mRNA level, suggesting translational regulation. Finally, overexpression of TPM1 in breast cancer MCF-7 cells suppressed anchorage-independent growth. Thus, down-regulation of TPM1 by mir-21 may explain, at least in part, why suppression of mir-21 can inhibit tumor growth, further supporting the notion that mir-21 functions as an oncogene. MicroRNAs (miRNAs) 2The abbreviations used are: miRNA, microRNA; siRNA, short interfering RNA; RT, reverse transcription; qRT, quantitative reverse transcription; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; TPM, tropomyosin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide; 5-aza-dC, 5-aza-2′-deoxycytidine; UTR, untranslated region; 2-DIGE, two-dimensional differentiation in-gel. are a class of naturally occurring small noncoding RNAs that regulate gene expression by targeting mRNAs for translational repression or cleavage (1Pillai R.S. RNA. 2005; 11: 1753-1761Crossref PubMed Scopus (604) Google Scholar, 2Zamore P.D. Haley B. Science. 2005; 309: 1519-1524Crossref PubMed Scopus (1132) Google Scholar). Like protein-coding mRNAs, miRNAs are transcribed as long primary transcripts in the nucleus. However, unlike protein-coding mRNAs, miRNAs are subsequently cleaved to produce stem-loop-structured precursor molecules of ∼70 nucleotides in length (pre-miRNAs) by the nuclear RNase III enzyme Drosha (3Kim V.N. Nat. Rev. Mol. Cell. Biol. 2005; 6: 376-385Crossref PubMed Scopus (2013) Google Scholar). The pre-miRNAs are then exported to the cytoplasm, where the RNase III enzyme Dicer further processes them into mature miRNAs (∼22 nucleotides). Thus, miRNAs are related to, but distinct from, short inferring RNAs (siRNAs) (4Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29863) Google Scholar, 5Fitzgerald K. Curr. Opin. Drug Discovery Dev. 2005; 8: 557-566PubMed Google Scholar). A key difference between siRNAs and miRNAs is that siRNAs require almost identical sequences to targets to exert their silencing function, whereas miRNAs bind through partial sequence homology to the 3′-untranslated region (3′-UTR) of target genes. Because of this unique feature, a single miRNA has multiple targets. Thus, miRNAs could regulate a large fraction of protein-coding genes, and as high as 30% of all genes could be miRNA targets (6Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9936) Google Scholar). As a new layer of gene regulation mechanism, miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation, and apoptosis (7Croce C.M. Calin G.A. Cell. 2005; 122: 6-7Abstract Full Text Full Text PDF PubMed Scopus (1221) Google Scholar, 8Chen C.Z. Li L. Lodish H.F. Bartel D.P. Science. 2004; 303: 83-86Crossref PubMed Scopus (2804) Google Scholar). Hence, deregulation of miRNA expression may lead to a variety of disorders. Aberrant expression of miRNAs in cancer has been well documented (7Croce C.M. Calin G.A. Cell. 2005; 122: 6-7Abstract Full Text Full Text PDF PubMed Scopus (1221) Google Scholar). Apparently, miRNAs may function as tumor suppressors or oncogenes by targeting oncogenes or tumor suppressor genes (9Chen C.Z. N. Engl. J. Med. 2005; 353: 1768-1771Crossref PubMed Scopus (693) Google Scholar). In this regard, tumor-suppressive miRNAs are usually underexpressed in tumors. For instance, let-7 is down-regulated in lung cancer (10Takamizawa J. Konishi H. Yanagisawa K. Tomida S. Osada H. Endoh H. Harano T. Yatabe Y. Nagino M. Nimura Y. Mitsudomi T. Takahashi T. Cancer Res. 2004; 64: 3753-3756Crossref PubMed Scopus (2164) Google Scholar, 11Johnson 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 (3119) Google Scholar). Furthermore, more than 60% of investigated patients suffering from B-cell chronic lymphocytic leukemia (B-CLL) have been reported to show a deletion at chromosome 13q14 where the mir-15 and mir-16 genes are located; these genes are under-represented in many B-CLL patients (12Calin G.A. Dumitru C.D. Shimizu M. Bichi R. Zupo S. Noch E. Aldler H. Rattan S. Keating M. Rai K. Rassenti L. Kipps T. Negrini M. Bullrich F. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15524-15529Crossref PubMed Scopus (4274) Google Scholar). Deregulation of miRNAs has also been reported in many other types of cancers. However, although miRNAs have been the subject of extensive research in recent years, the molecular basis of miRNA-mediated gene regulation and the effect of these genes on tumor growth remain largely unknown because of our limited understanding of miRNA target genes. Identification of miRNA target genes has been a great challenge. Computational algorithms have been the major driving force in predicting miRNA targets (13Stark A. Brennecke J. Russell R.B. Cohen S.M. PLoS Biol. 2003; 1: e60Crossref PubMed Scopus (628) Google Scholar, 14Lewis B.P. Shih I.H. Jones-Rhoades M.W. Bartel D.P. Burge C.B. Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4250) Google Scholar, 15Kiriakidou M. Nelson P.T. Kouranov A. Fitziev P. Bouyioukos C. Mourelatos Z. Hatzigeorgiou A. Genes Dev. 2004; 18: 1165-1178Crossref PubMed Scopus (644) Google Scholar). The approaches are mainly based on base pairing of miRNA and target gene 3′-UTR, emphasizing the location of miRNA complementary elements in 3′-UTR of target mRNAs, the concentration in the seed (6-8 bp) of continuous Watson-Crick base pairing in the 5′ proximal half of the miRNA, and the phylogenetic conservation of the complementary sequences in 3′-UTRs of orthologous genes. However, evidence suggests that perfect seed pairing may not necessarily be a reliable predictor for miRNA-target interactions (16Didiano D. Hobert O. Nat. Struct. Mol. Biol. 2006; 13: 849-851Crossref PubMed Scopus (354) Google Scholar), which may explain why many predicted target sites are nonfunctional. A recent study also suggests that there may be at least three types of miRNA-mRNA interactions in mammals (17Smalheiser N.R. Torvik V.I. Methods Mol. Biol. 2006; 342: 115-127PubMed Google Scholar). Hence, with few exceptions, large portion of the physiologic targets for miRNAs remain to be identified or verified experimentally. In this study, we analyzed tumors derived from breast cancer MCF-7 cells treated with antisense mir-21 oligonucleotide (anti-mir-21) or the negative control by two-dimensional differentiation in-gel (2-DIGE) and identified the tumor suppressor tropomyosin 1 (TPM1) as a putative mir-21 target. Subsequent experiments confirmed that mir-21 down-regulated expression of TPM1, whereas anti-mir-21 up-regulated its expression through the mir-21 binding site at the 3′-UTR region. Furthermore, ectopic expression of TPM1 suppressed anchorage-independent growth. Cell Culture—MCF-7 cells (obtained from American Type Cell Collection, Manassas, VA) were grown in RPMI 1640 (Cambrex, Walkersville, MD) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mm glutamine, 100 units of penicillin/ml, and 100 μg of streptomycin/ml (Cambrex). MCF10A cells (ATCC) were grown in serum-free mammary epithelial growth medium (from Cambrex) supplemented with 100 ng/ml cholera toxin (EMD Biosciences, San Diego, CA). 293T cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (Cambrex) supplemented with 10% fetal bovine serum. All cells were incubated at 37 °C in a humidified chamber supplemented with and the negative control were from was from CA). of MCF-7 cells was performed with the the cells were in at 30% on the μg of or control was used for in medium of 293T cells was performed the as Cell Res. PubMed Scopus Google Scholar). The negative control oligonucleotide or anti-mir-21 oligonucleotide from at or μg of was used for was by or of mir-21 by miRNA used the C. Z. M. Res. 2005; PubMed Scopus Google Scholar, K. C. Res. 2006; PubMed Scopus Google to the expression level of mature For RNA was used in and with the The was the °C for °C for °C for and then on the the were at and of the was used for with The was at °C for by of °C for and °C for in the real-time The real-time were analyzed and as miRNA expression of which was then to C. Z. M. Res. 2005; PubMed Scopus Google Scholar). The and for mir-21 C. Z. M. Res. 2005; PubMed Scopus Google were from or RNA was used for of TPM1 of TPM1 was performed the the °C for by of °C for °C for 1 and °C for were and were from a mir-21, we modified by the by then a from MCF10A where the site is and where the site is The was cloned into and was subsequently cloned into this modified at the and TPM1 plus 3′-UTR was from MCF-7 cells and then cloned into and into the The 3′-UTR region of TPM1 was also from MCF-7 cells and the and was cloned into control at the a the protein we also used and as indicated was cloned into at the and sites with the region. the 3′-UTR of TPM1 into a which was from the we modified the by a in the of the multiple sites by and then the into site of this modified All were verified by into the cells were in and with luciferase the as the cells were into and for luciferase a luciferase to the was used for Cell with control or the cells were into at The was used to cell growth as S. H. Z. F. in 2006; Google Scholar). anchorage-independent growth of the cells were grown in to a S. J. M. PubMed Google Scholar). 1 with TPM1, cells were and with medium to in a concentration of of this cell were in with in medium and at 37 °C with cell in cell was by in CA). Western protein was from tumor or 293T cells with an in cell mm 100 mm 1 mm concentration was the protein The was with or and then with were with or were the Biosciences, were from and were in the of All studies were in with of and a by the MCF-7 cells were with at and into mammary of tumor growth, a of was the usually 1 anti-mir-21 or negative control oligonucleotide was to tumor sites by of the oligonucleotide of of the was was other of MCF-7 tumors were and in a °C of that were and at °C were for and a by CA). protein was and with or in the was at and in the was in were and to are as and is as by of by have that of MCF-7 cells with anti-mir-21 tumor growth in a S. H. Z. F. in 2006; Google Scholar). 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Because miRNAs are to regulate gene expression mainly through translational repression in we to the expression of from the tumor with was from tumors derived from MCF-7 cells treated with the negative control with or anti-mir-21 with two-dimensional in which are in this with fluorescent in a single and for of expression the by and we found that were up-regulated or down-regulated as by or green in is in agreement with the that also or down-regulation of many J. N. R. T. M. M. 2005; PubMed Scopus Google Scholar), because of these may be to the effect of miRNA regulation. of of tumor from revealed an almost identical to that of suggesting the of this are in up-regulated by anti-mir-21 because are potential targets for protein that were up-regulated more than in the tumor treated with anti-mir-21 compared with the negative these are in identified of them with a three have been in TPM1 N. M. Mol. Cell. Biol. PubMed Scopus Google Scholar), protein F. F. S. M. E. F. A. S. Cancer Res. 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TPM1 has variants through TPM1 variants 1 and a putative mir-21 binding as predicted by the miRNA base target 1 from in a sequence for and also by an nucleotides of the 3′-UTR The potential base pairing between mir-21 and TPM1 3′-UTR is in Thus, we to this region of both variants from MCF-7 to TPM1 Hence, we cloned this 1 3′-UTR into control As in the luciferase in 293T cells for was than that of control suggesting that TPM1 3′-UTR a that this region is mir-21 we 293T cells with the with the or the The ectopic expression of mir-21 was confirmed by real-time which revealed a mir-21 expression in the cells than in control In anti-mir-21 mir-21 by almost as by the then the 293T cells with of As in of luciferase by mir-21 was suggesting that this regulation is to In mir-21 no effect on which is derived from and the mir-21 binding site In we the effect of anti-mir-21 on the luciferase of As mir-21 suppressed the luciferase activity, whereas anti-mir-21 the luciferase further suggesting that expression of TPM1 is regulated by the of the mir-21 binding site in its we the mir-21 binding site in 1 As in mir-21 anti-mir-21 effect on the luciferase activity, the of this mir-21 binding of TPM1 expression by mir-21 at the translational expression of in 293T cells as by Western The was with and then with as The effect of mir-21 or anti-mir-21 on the protein of and or and in which the mir-21 binding site was is and are of at least three and are the of three experiments mir-21 or anti-mir-21 has no effect on the mRNA of as by real-time negative control that down-regulation of luciferase by mir-21 was not to the reporter we we reporter in In this we cloned into the site of the modified with the luciferase the level of was by mir-21 but was by as by Western blot or mir-21 TPM1 at the repression is a major of miRNAs to regulate gene expression 2006; PubMed Scopus Google Scholar). mir-21 also TPM1 through translational we cloned the TPM1 plus the 3′-UTR into of TPM1 was confirmed by although ectopic expression of mir-21 TPM1 anti-mir-21 enhanced TPM1 protein and further the of the mir-21 binding we experiments with in which the site was of this site abolished the effect of mir-21 or anti-mir-21 on TPM1 expression at the protein level and However, the effect of mir-21 or anti-mir-21 on TPM1 at the protein level, no effect on the TPM1 mRNA level was by real-time for these that the mir-21 binding site in the region is for regulation at the translational In we a with the TPM1 plus the 3′-UTR confirmed its expression of its protein by Western blot a of we the green protein all the cell for protein was to the Moreover, mir-21 also the protein as by which was confirmed by Western blot In this we the cells with and a between with and with Thus, the mir-21 binding site is from the by TPM1 it is further that TPM1 is a mir-21 of expression of the protein by expression of or in 293T The and were into 293T cells were on and grown for were with and a that protein is in the as compared with which is the and effect of mir-21 on expression of the protein as by or Western blot with indicated a between with control and with of TPM1 Cell in and previous studies have indicated that suppression of TPM1 is a of many and TPM1 functions as a tumor suppressor S. 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Full Text PDF PubMed Scopus Google Scholar), although the 3′-UTR of may not be to tumor suppression or may not be for tumor suppression in other types of cells Mol. Biol. Cell. 8: PubMed Scopus Google Scholar, 13: Google Scholar). it be of to miRNAs as mir-21 with this region. this we that overexpression of this 3′-UTR region a of miRNAs in the to tumor In TPM1 expression can be regulated by study our the regulation of TPM1, a tumor suppressor Thus, in to as TPM1 is also regulated at the translational level by that a single miRNA has multiple we that mir-21 also has many targets. is our that more mir-21 targets be identified in the with the that we be to the molecular basis of