miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27Kip1. We recognize two target sites for the microRNAs in the 3′ untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G1 to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27Kip1 in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27Kip1, and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27Kip1 down-regulation. MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27Kip1. We recognize two target sites for the microRNAs in the 3′ untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G1 to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27Kip1 in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27Kip1, and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27Kip1 down-regulation. MicroRNAs (miRNAs) 5The abbreviations used are: miRNA, microRNA; UTR, untranslated region; siRNA, small interfering RNA; PCa, prostate cancer; MTS, 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium, inner salt. are a wide class of small, noncoding RNAs that negatively regulate protein expression at the post-transcriptional level. Through the specific targeting of the 3′ UTRs of multicellular eukaryotic mRNAs, miRNAs down-regulate gene expression by either inducing degradation of target mRNAs or impairing their translation (1Kim V.N. Mol. Cell. 2005; 19: 1-15Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 2Petersen C.P. Bordeleau M.E. Pelletier J. Sharp P.A. Mol. Cell. 2006; 21: 533-542Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). The expression of many microRNAs was shown to be temporally and spatially regulated, whereas the disruption of their physiological expression patterns was associated with several examples of human tumorigenesis, suggesting that they may play a role as a novel class of oncogenes or tumor suppressor genes (3Gregory R.I. Shiekhattar R. Cancer Res. 2005; 65: 3509-3512Crossref PubMed Scopus (572) Google Scholar). In fact, single or small sets of microRNAs were demonstrated to be dysregulated in diverse cancer subtypes including Burkitt lymphoma (4Metzler M. Wilda M. Busch K. Viehmann S. Borkhardt A. Genes Chromosomes Cancer. 2004; 39: 167-169Crossref PubMed Scopus (503) Google Scholar), colorectal cancer (5Michael M.Z. O'Connor S.M. van Holst Pellekaan N.G. Young G.P. James R.J. Mol. Cancer Res. 2003; 12: 882-891Google Scholar), lung cancer (6Takamizawa 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), breast cancer (7Iorio M.V. Ferracin M. Liu C.G. Veronese A. Spizzo R. Sabbioni S. Magri E. Pedriali M. Fabbri M. Campiglio M. Menare S. Palazzo J.P. Rosenberg A. Musiani P. Volinia S. Nenci I. Calin G.A. Querzoli P. Negrini M. Croce C.M. Cancer Res. 2005; 65: 7065-7070Crossref PubMed Scopus (3498) Google Scholar), papillary thyroid carcinoma (8He H. Jazdzewski K. Li W. Liyanarachchi S. Nagy R. Volinia S. Calin G.A. Liu C.G. Franssila K. Suster S. Kloos R.T. Croce C.M. de la Chappelle A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 19075-19080Crossref PubMed Scopus (1075) Google Scholar), hepatocellular carcinoma (9Murakami Y. Yasuda T. Saigo K. Urashima T. Toyoda H. Okanoue T. Shimotohno K. Oncogene. 2006; 25: 2537-2545Crossref PubMed Scopus (1037) Google Scholar), and glioblastoma (10Chan J.A. Cancer Res. 2005; 65: 6029-6033Crossref PubMed Scopus (2240) Google Scholar, 11Ciafre S.A. Galardi S. Mangiola A. Ferracin M. Liu C-G. Sabatino G. Negrini M. Maira G. Croce C.M. Farace M.G Biochem. Biophys. Res. Commun. 2005; 334: 1351-1358Crossref PubMed Scopus (953) Google Scholar). The general rule stemming from these studies is that the non-physiological modulation of micro-RNA expression frequently characterizes cancer, thus making the comprehension of microRNA expression an important goal for diagnostic and prognostic applications, especially when this knowledge is further strengthened by the discovery of the molecular targets specifically modulated by microRNAs. Prostate cancer, the most common malignant disease in the Western world, causes about 80,000 deaths a year in Europe (12Bracarda S. Crit. Rev. Oncol. Hematol. 2005; 56: 379-396Crossref PubMed Scopus (90) Google Scholar). Despite considerable efforts made in recent years to understand prostate tumorigenesis, the molecular mechanisms involved in its initiation and progression remain largely unknown. Among factors whose misregulation was tightly linked to prostate cancer (PCa) progression, the cyclin-dependent kinase inhibitor p27Kip1 is a well established marker of poor prognosis as it was shown that absent or decreased p27Kip1 expression is associated with high tumor grade and poor prognosis of PCa and of several other human cancers (13Tsihlias J. Kapusta L. Slingerland J. Annu. Rev. Med. 1999; 50: 401-423Crossref PubMed Scopus (291) Google Scholar, 14Macri E. Loda M. Cancer Metastasis Rev. 1998; 17: 337-344Crossref PubMed Scopus (67) Google Scholar, 15Lloyd R.V. Erickson L.A. Jin L. Kulig E. Qian X. Cheville J.C. Scheithauer B.W. Am. J. Pathol. 1999; 154: 313-323Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 16Cheville J.C. Lloyd R.V. Sebo T.J. Cheng L. Erickson L. Bostwick D.G. Lohse C.M. Wollan P. Mod. Pathos. 1998; 11: 324-328PubMed Google Scholar, 17Tsihlias J. Kapusta L.R. DeBoer G. Morava-Protzner I. Zbieranowski I. Bhattacharya N. Catzavelos G.C. Klotz L.H. Slingerland J.M. Cancer Res. 1998; 58: 542-548PubMed Google Scholar, 18Yang R.M. Naitoh J. Murphy M. Wang H.J. Phillipson J. deKernion J.B. Loda M. Reiter R.E. J. Urol. 1998; 159: 941-945Crossref PubMed Scopus (277) Google Scholar). It is also well known that p27Kip1 regulation, both in physiological and pathological conditions, is exerted mostly at a post-transcriptional level (19Belletti B. Nicoloso M.S. Schiappacassi M. Chimienti E. Berton S. Lovat F. Colombatti A. Baldassarre G. Curr. Med. Chem. 2005; 12: 1589-1605Crossref PubMed Scopus (77) Google Scholar). In this work we describe the differential expression of two microRNAs, miR-221 and miR-222, encoded in tandem from a gene cluster located on chromosome X, in three human prostate carcinoma cell lines, the androgen-independent, strongly aggressive PC3 cell line, the androgen-responsive 22Rv1, and the androgen dependent, slowly growing LNCaP, which represent models of distinct stages of prostate carcinoma progression. Consistently with the proposed role of microRNAs as regulators of key components of cell cycle progression, here we identify p27Kip1 as a target for miR-221/miR-222. We show that p27Kip1 expression in the three PCa cell lines inversely correlates with that of miR-221/miR-222, and that the ectopic overexpression of miR-221 or both microRNAs in LNCaP, where they are normally almost undetectable, has deep consequences on the proliferation rate and the cell cycle phase distribution. We propose that p27Kip1 is an important functional target for miR-221/222 in prostate carcinoma, and that the modulation of these microRNAs might be used as a molecular marker to characterize the progression of this tumor. Cell Lines and Transfections—All cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 20 mm l-glutamine, 100 units/ml of penicillin G sodium, and 100 μg/ml streptomycin sulfate in a humidified atmosphere containing 5% CO2 at 37 °C. Transfections were performed by Lipofectamine 2000 reagent (Invitrogen) using 8 μg of plasmid DNA in Opti-MEM I (Invitrogen), as recommended by the manufacturer. For transient transfections, 7 μg of pCDNA(+)3.1-based plasmids were co-transfected with a 1:7 relative amount of a reporter plasmid, pEGFP-C3 (Clontech), to monitor transfection efficiency. Cells were analyzed by fluorescence microscopy 48 h after transfection to calculate the transfection efficiency. For each well, the cell number in four random microscopic fields was counted and transfection efficiency for each sample was estimated as the mean value of green fluorescent protein expressing cells over 100 cells per field. When establishing stable transfectants, the transfected cells were selected by adding 0.4 to the of 3′ of the human gene was using and and of the in to the was used to by inverse the plasmid LNCaP cells were transfected by Lipofectamine 2000 (Invitrogen) with or plasmid Cells were 48 h and with to the were performed in miR-222, and cluster were by from human DNA using the miR-221 miR-221 miR-222 miR-222 The of were by and the of (Invitrogen) for miR-222, and sites for miR-221 and expression was by after 48 h from transfection in LNCaP and cells and p27 LNA oligonucleotides miR-221 and miR-222 were from were transfected by Lipofectamine 2000 (Invitrogen) PC3 cells at a of the cells were and miRNAs and p27 protein 7 were for at in p27 were from and transfected by Lipofectamine 2000 LNCaP cells at a of 100 Cell was by using Cell with in which cells a were in a and for h to to the of the The cell growth was at 48 and h after the cellular the 20 of were to 100 of and for h at 37 and the was at were performed in of miR-222 LNCaP Cells and Cell were in a for h to to the of the well, and transfected with or using h after this cells were transfected with the of or siRNA, with 100 miR-222 or a in this in to the transfection with miRNA, and cell growth was by from that at and growth was by soft as LNCaP or PC3 cells per were in on of a containing agar. In the or or LNA cells were h after were at 37 at 5% CO2 in a humidified for and with for mm LNCaP or mm PC3 in were counted a microscopic at was performed in on two and was from LNCaP, and cells with reagent (Invitrogen) to the For of μg of were on 10% and to The specific with kinase in the of miR-222, and were with or transfected cells were in and at 37 for 48 Cells were with for with and for at with to DNA in cells were with μg/ml for h at cells were in of and with of for in the cells were and in containing μg/ml for on a using For cells were transfected and at 37 °C. cells were and in in and with μg/ml and μg/ml for at 37 °C. were performed each of three μg of protein was on and to The of p27 expression were by using the or the a expression were by The or was using Western were with or MicroRNAs miR-221 and miR-222 in PC3 Cells in LNCaP and 22Rv1 and to of goal was to for microRNAs in prostate this we three human PCa cell lines, LNCaP, and 22Rv1, three stages of prostate carcinoma progression. performed by that the microRNAs miR-221 and miR-222 show a differential they are in PC3 cells, from a of an androgen-independent, aggressive whereas they are almost absent in LNCaP, from a of an slowly growing carcinoma The expression in the 22Rv1 cell line, an androgen-responsive cell line, was undetectable, in LNCaP We performed a C.P. S. Res. 2006; PubMed Scopus Google for target mRNAs of both and we that the 3′ of human two sites by the miR-221 and miR-222 In fact, the of these two miRNAs are and target the located at and of the miR-222 and miR-221 is at in the 3′ of p27 mRNA, where it is also associated with the relationship inversely p27Kip1 and miR-221 and miR-222, we by p27 expression in the three prostate carcinoma cell Western that p27 is in LNCaP and 22Rv1, whereas it is strongly in PC3 cells The of these results a that the expression of miR-221 and miR-222 might be one of the mechanisms to negatively regulate p27Kip1 in prostate carcinoma cells. The of miR-222, or p27Kip1 in LNCaP and 22Rv1 Cells and the of the MicroRNAs p27Kip1 in PC3 these microRNAs p27Kip1 expression in LNCaP cellular we analyzed the consequences of the ectopic expression of miR-221 and We made three and miR-222, or the region with miR-221 and miR-222 encoded in the of that LNCaP cells with or high of the microRNAs, whereas expression was in cells. results were with the 22Rv1 cell line was in all of the miRNAs in the cells. Western performed on the cells that the p27Kip1 protein was in both LNCaP and 22Rv1 cells expressing the single miRNAs or a of as compared with cells transfected with the strongly the that miR-221 and miR-222 regulate p27Kip1 expression in LNCaP and 22Rv1 cells. the of these miRNAs in PC3 cells, where they are normally we miR-221 or miR-222 we transfected PC3 cells with LNA antisense oligonucleotides targeting either miR-221 or miR-222, and we analyzed the on p27 PC3 cells transfected with and LNA a in miR-221 and miR-222 when compared with cells transfected with LNA a microRNA in these cells the of miR-221 or miR-222 was by an of p27 protein of about The results that we by both the overexpression and the of miR-221 miR-222 the of p27 as an target of in LNCaP, 22Rv1, and PC3 PCa cell The of the 3′ of p27 of a to miR-221 and show that the 3′ of p27 sites for the with miR-221 and miR-222 and is for the of these miRNAs on p27 we the 3′ region of the and we used this reporter to LNCaP cells. The reporter was transfected LNCaP cells transfected with plasmids or that the of miR-222, or miR-221 and miR-222 in tandem strongly expression as relative The of was in all transfected cell lines, either expressing miR-221 or miR-222, or a of miR-221 and the when we as a reporter a plasmid the 3′ of p27 where two sites for miR-221 and miR-222 were by we a on compared with LNCaP cells that results the the 3′ of p27 as a target for miR-221 and miR-222, and that the two sites thus strongly to the the post-transcriptional of the The of miR-221/222 of LNCaP Cells and a to the S of Cell is known to play a key role as a of cell cycle progression, strongly We the cell growth potential of transfected LNCaP cells expressing miR-222, or both miR-221 and miR-222 was as a of the demonstrated p27 the results of an where cell of LNCaP cells is compared with that of the the expression of the microRNAs a in growth rate at h after the of the both in the of miR-221 or miR-222 or the two microRNAs in results a in a we performed to show that a of p27 by from microRNA expression to when we transfected LNCaP cells with that were to p27 of about we a in cell as compared with cells p27 in LNCaP cells, either by miR-221/222 expression or by is sufficient to induce a cell growth strongly the p27 of these of we a where we the proliferation by microRNAs in LNCaP cells transfected with The of this was to and the cellular to miR-221 or miR-222 When one of the microRNAs, miR-222, was transfected LNCaP cells with we after 48 h from miR-222 and miR-222 to to the of p27 protein and to the growth is at when the functional consequences of p27 are We LNCaP cell cycle phase through that LNCaP cells expressing miR-222, or the miR-221/222 cluster a in S phase as compared with cells, with a of the G1 for cell growth cell cycle phase was also by cells from G1 S phase results demonstrate the of miR-221 and miR-222 to a cell cycle and the role of these two miRNAs as regulators of p27Kip1. The of miR-221 or miR-222 the of LNCaP and miR-221/222 It in PC3 of cellular is the of tumor cells to in an in a the of expression on this cell we the of transfected LNCaP cells to and to when at in soft agar. shown in and LNCaP cells expressing miR-221 or miR-222 many the cells for and for results that miR-221 and miR-222 expression affects of tumor cell In we compared the of miR-221 and miR-222 expression with that of on the potential of LNCaP cells. The by LNCaP cells with were those by LNCaP cells with a and as for proliferation we the of results in both of which of small was used to target we also that the of miR-221 and miR-222 through LNA antisense oligonucleotides in PC3 cells was to strongly the number of the growing in soft and in with about the of p27 after transfection with LNA that directly miR-221 and miR-222 to p27 and to the in cell the of In the an growing number of a between several of human cancer and the expression of microRNAs a A. Rev. Cancer. 2006; PubMed Scopus Google Scholar). Despite the of work that has to in a of the were the targets of the microRNAs that were shown to be specifically modulated in it is that comprehension of the functional role of microRNAs in oncogenesis be by their of in each of in this work we on a of microRNAs, miR-221 and miR-222, overexpressed in the specific of prostate The we here are on that this of miRNAs are in a PCa cell line from a strongly aggressive tumor other cell lines from slowly growing and these PCa cell lines are as models of stages of PCa progression S. PubMed Scopus Google Scholar), we that the expression of miR-221/222 in PC3 aggressive cells might be a marker of their is strengthened by that one and modulated by miR-221/222 in PCa cell lines, is the cell cycle inhibitor p27Kip1, in the family of tumor Through the we demonstrated that at two sites are in the 3′ we that miR-221 and miR-222 are inversely linked to those of p27 expression in 22Rv1, and in LNCaP cell the ectopic expression of these miRNAs or their knock-down are to induce the on p27 expression in LNCaP, 22Rv1, and PC3 cells. We functional about the role of miR-221 and miR-222 in PCa, by that the expression of these microRNAs is per to induce an enhancement of LNCaP cell growth which is and to that by a specific of In this in cell growth is tightly linked to the G1 to S shift we in the cells, which is in with modulation of a known of the cell cycle is by their to in an a to the of PCa cells. The potential was strongly when miR-221 or miR-222 were overexpressed in LNCaP cells, and was in PC3 cells where miR-221 and miR-222 were through LNA thus with a further about the of this microRNA in PCa The inverse between the inhibitor p27 and prognosis in several human including prostate carcinoma, is well known E. Loda M. Cancer Metastasis Rev. 1998; 17: 337-344Crossref PubMed Scopus (67) Google Scholar, J. M. J. Cell. PubMed Scopus Google Scholar). p27 is an in prostate carcinoma as it is frequently in which is as a carcinoma Am. J. Pathol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The that p27 is a tumor suppressor is of for the of as it be in with the regulatory of microRNAs by the amount of their We that this regulatory exerted by miR-221/222 on the of p27 protein in the cell might be as of the made of several post-transcriptional mechanisms a of p27 amount to and at in one by that describe an modulation of miR-221 miR-222 in a of the of which is also known to be by p27 or miR-221 and miR-222 are overexpressed in of the and S. Calin G.A. Liu C.G. S. A. F. R. M. Ferracin M. N. G. A. A. Negrini M. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar, Y. J. J. Cancer. PubMed Scopus Google Scholar), and are strongly in papillary thyroid carcinoma (8He H. Jazdzewski K. Li W. Liyanarachchi S. Nagy R. Volinia S. Calin G.A. Liu C.G. Franssila K. Suster S. Kloos R.T. Croce C.M. de la Chappelle A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 19075-19080Crossref PubMed Scopus (1075) Google Scholar). miR-222 is a marker of hepatocellular carcinoma well of the overexpressed about in the (9Murakami Y. Yasuda T. Saigo K. Urashima T. Toyoda H. Okanoue T. Shimotohno K. Oncogene. 2006; 25: 2537-2545Crossref PubMed Scopus (1037) Google Scholar). miR-221 and miR-222 was also in a as associated with important of poor prognosis G.A. Ferracin M. A. G. M. M.V. R. Fabbri M. R. T. F. R. L. H. Volinia S. Liu C.G. T.J. Negrini M. Croce C.M. N. J. Med. 2005; PubMed Scopus Google Scholar). We demonstrated that miR-221 and miR-222 are the most microRNAs in glioblastoma S.A. Galardi S. Mangiola A. Ferracin M. Liu C-G. Sabatino G. Negrini M. Maira G. Croce C.M. Farace M.G Biochem. Biophys. Res. Commun. 2005; 334: 1351-1358Crossref PubMed Scopus (953) Google Scholar), and we also that suggest the of a between miR-221/222 overexpression and p27 down-regulation in this model of human tumor. S. A. and M. In three was the modulation of miR-221 and miR-222 in with the of their which was the kinase in one miR-221 and miR-222 to be in an cell line with a overexpression of N. L. E. R. F. F. S. M. Calin G.A. Liu C.G. A. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar), whereas in the other the two miRNAs in tandem were overexpressed in papillary thyroid carcinoma, a down-regulation of (8He H. Jazdzewski K. Li W. Liyanarachchi S. Nagy R. Volinia S. Calin G.A. Liu C.G. Franssila K. Suster S. Kloos R.T. Croce C.M. de la Chappelle A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 19075-19080Crossref PubMed Scopus (1075) Google Scholar). In a the overexpression of miR-221 and miR-222 in their to the of cell by targeting its L. A. L. M. L. K. A. S. G. 2006; PubMed Scopus Google Scholar), suggesting a role for miR-221 and miR-222 as of the of new in physiological and pathological We that which identify p27Kip1 as a target for miR-221 and miR-222 in the of prostate carcinoma cell lines, a of the of gene it is well known and that the relationship between microRNAs and target mRNAs is a to as the can be by one miRNA, and that the of many and which miRNAs target one 3′ is strongly by the specific cellular 2005; PubMed Scopus Google Scholar). that targets in the of cell proliferation may as a tumor suppressor in cancers and as an in on which targets are in that specific cellular In results suggest that overexpression of miR-221 and miR-222 may to the growth and progression of prostate carcinoma, at in by p27 functional studies are for the comprehension of the molecular of the of this carcinoma, and new to targeting specific tumor as the overexpressed microRNAs. We are to and for and We are to for the