ST14 (Suppression of Tumorigenicity 14) Gene Is a Target for miR-27b, and the Inhibitory Effect of ST14 on Cell Growth Is Independent of miR-27b Regulation
2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês
10.1074/jbc.m109.012617
ISSN1083-351X
AutoresYanfang Wang, Rajamani Rathinam, Amelia Walch, Suresh K. Alahari,
Tópico(s)RNA Research and Splicing
ResumoMicroRNAs are noncoding, endogenous small RNAs that regulate target genes by cleavage of the targeted mRNA or translational repression. We investigated the microRNAome using 2-color microarrays in a highly invasive human breast cancer cell line, MDA-MB-231 (subline 4175) and a noninvasive breast epithelial cell line, MCF10A. We found 13 microRNAs that were up-regulated, and nine were down-regulated significantly in 4175 cells (p < 0.05, -fold change >2) compared with MCF10A cells. Interestingly, miR-27b and its putative target gene, ST14 (suppressor of tumorigenicity 14), had inverse expression pattern in breast cancer cells. The 3′-untranslated region of ST14 contains a regulatory element for miR-27b, and our luciferase experiments indicate that antisense miR-27b enhances ST14 expression in cancer cells. Furthermore, antagomir of miR-27b suppressed cell invasion in 4175 cells, whereas pre-miR-27b stimulated invasion in moderately invasive ZR75 breast cancer cells. In addition, ST14 reduces cell proliferation as well as cell migration and invasion. Analysis of human breast tumors revealed that miR-27b expression increases during cancer progression, paralleling a decrease in ST14 expression. Furthermore, our data indicate that ST14 inhibits cells from entering into S phase by up-regulating p27, which results in down-regulation of cyclin E-CDK2 complexes, suggesting ST14 reduces cell growth through its effects on cell cycle-related proteins. Introduction of miR-27b into ST14-expressing cells did not suppress the effect on cell growth. These findings suggest that ST14 plays an important role in several biological processes, and some effects are not completely dependent on miR-27b regulation. MicroRNAs are noncoding, endogenous small RNAs that regulate target genes by cleavage of the targeted mRNA or translational repression. We investigated the microRNAome using 2-color microarrays in a highly invasive human breast cancer cell line, MDA-MB-231 (subline 4175) and a noninvasive breast epithelial cell line, MCF10A. We found 13 microRNAs that were up-regulated, and nine were down-regulated significantly in 4175 cells (p < 0.05, -fold change >2) compared with MCF10A cells. Interestingly, miR-27b and its putative target gene, ST14 (suppressor of tumorigenicity 14), had inverse expression pattern in breast cancer cells. The 3′-untranslated region of ST14 contains a regulatory element for miR-27b, and our luciferase experiments indicate that antisense miR-27b enhances ST14 expression in cancer cells. Furthermore, antagomir of miR-27b suppressed cell invasion in 4175 cells, whereas pre-miR-27b stimulated invasion in moderately invasive ZR75 breast cancer cells. In addition, ST14 reduces cell proliferation as well as cell migration and invasion. Analysis of human breast tumors revealed that miR-27b expression increases during cancer progression, paralleling a decrease in ST14 expression. Furthermore, our data indicate that ST14 inhibits cells from entering into S phase by up-regulating p27, which results in down-regulation of cyclin E-CDK2 complexes, suggesting ST14 reduces cell growth through its effects on cell cycle-related proteins. Introduction of miR-27b into ST14-expressing cells did not suppress the effect on cell growth. These findings suggest that ST14 plays an important role in several biological processes, and some effects are not completely dependent on miR-27b regulation. MicroRNAs (miRNAs) 2The abbreviations used are: miRNAmicroRNAsUTRuntranslated regionRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseQPCRreal time quantitative PCRX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideIDCinvasive ductal carcinomaCDKcyclin-dependent kinasepRbretinoblastoma protein. 2The abbreviations used are: miRNAmicroRNAsUTRuntranslated regionRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseQPCRreal time quantitative PCRX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideIDCinvasive ductal carcinomaCDKcyclin-dependent kinasepRbretinoblastoma protein. are a new class of noncoding, endogenous, small RNAs and are highly conserved between species (1Ambros V. Nature. 2004; 431: 350-355Crossref PubMed Scopus (8911) Google Scholar). miRNAs are generated by the RNase III type enzyme Dicer from premature miRNAs, which are endogenous, hairpin-shaped secondary transcripts (2Lee Y. Jeon K. Lee J.T. Kim S. Kim V.N. EMBO J. 2002; 21: 4663-4670Crossref PubMed Scopus (1671) Google Scholar, 3Lee Y. Ahn C. Han J. Choi H. Kim J. Yim J. Lee J. Provost P. Rådmark O. Kim S. Kim V.N. Nature. 2003; 425: 415-419Crossref PubMed Scopus (3919) Google Scholar). Mature miRNAs are single-stranded and 19–25 nucleotides in length. They bind to target sequences, usually at the 3′-UTR of genes, to down-regulate expression by cleavage of the mRNA and/or translational repression (4Ambros V. Cell. 2001; 107: 823-826Abstract Full Text Full Text PDF PubMed Scopus (1415) Google Scholar, 5He L. Hannon G.J. Nat. Rev. Genet. 2004; 5: 522-531Crossref PubMed Scopus (5551) Google Scholar). miRNA target genes can regulate cell development and differentiation, the cell cycle, and apoptosis (6Miska E.A. Curr. Opin. Genet. Dev. 2005; 15: 563-568Crossref PubMed Scopus (707) Google Scholar), and miRNAs therefore play an important regulatory role in cell biology.miRNAs may function as regulatory molecules and act as oncogenes or tumor suppressors (7Zhang B. Pan X. Cobb G.P. Anderson T.A. Dev. Biol. 2007; 302: 1-12Crossref PubMed Scopus (2136) Google Scholar). Jiang et al. analyzed the expression levels of 222 pre-miRNAs in 32 human cancer cell lines by real time PCR (8Jiang J. Lee E.J. Gusev Y. Schmittgen T.D. Nucleic Acids Res. 2005; 33: 5394-5403Crossref PubMed Scopus (447) Google Scholar). Expression profiling of mature miRNAs has been performed in many cancers, such as lung cancer (9Yanaihara N. Caplen N. Bowman E. Seike M. Kumamoto K. Yi M. Stephens R.M. Okamoto A. Yokota J. Tanaka T. Calin G.A. Liu C.G. Croce C.M. Harris C.C. Cancer Cell. 2006; 9: 189-198Abstract Full Text Full Text PDF PubMed Scopus (2675) Google Scholar), ovarian cancer (10Iorio M.V. Visone R. Di Leva G. Donati V. Petrocca F. Casalini P. Taccioli C. Volinia S. Liu C.G. Alder H. Calin G.A. Menard S. Croce C.M. Cancer Res. 2007; 67: 8699-8707Crossref PubMed Scopus (1287) Google Scholar), cervical cancer (11Lui W.O. Pourmand N. Patterson B.K. Fire A. Cancer Res. 2007; 67: 6031-6043Crossref PubMed Scopus (400) Google Scholar), prostate cancer (12Porkka K.P. Pfeiffer M.J. Waltering K.K. Vessella R.L. Tammela T.L. Visakorpi T. Cancer Res. 2007; 67: 6130-6135Crossref PubMed Scopus (785) Google Scholar), breast cancer (13Iorio M.V. Ferracin M. Liu C.G. Veronese A. Spizzo R. Sabbioni S. Magri E. Pedriali M. Fabbri M. Campiglio M. Ménard 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 (3437) Google Scholar), colon cancer (14Schetter A.J. Leung S.Y. Sohn J.J. Zanetti K.A. Bowman E.D. Yanaihara N. Yuen S.T. Chan T.L. Kwong D.L. Au G.K. Liu C.G. Calin G.A. Croce C.M. Harris C.C. JAMA. 2008; 299: 425-436Crossref PubMed Scopus (1421) Google Scholar), and head and neck cancer cell lines (15Tran N. McLean T. Zhang X. Zhao C.J. Thomson J.M. O'Brien C. Rose B. Biochem. Biophys. Res. Commun. 2007; 358: 12-17Crossref PubMed Scopus (226) Google Scholar). Several studies revealed specific miRNA expression patterns in normal and tumor samples (13Iorio M.V. Ferracin M. Liu C.G. Veronese A. Spizzo R. Sabbioni S. Magri E. Pedriali M. Fabbri M. Campiglio M. Ménard 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 (3437) Google Scholar, 16Budhu A. Jia H.L. Forgues M. Liu C.G. Goldstein D. Lam A. Zanetti K.A. Ye Q.H. Qin L.X. Croce C.M. Tang Z.Y. Wang X.W. Hepatology. 2008; 47: 897-907Crossref PubMed Scopus (621) Google Scholar). Lorio et al. (13Iorio M.V. Ferracin M. Liu C.G. Veronese A. Spizzo R. Sabbioni S. Magri E. Pedriali M. Fabbri M. Campiglio M. Ménard 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 (3437) Google Scholar) identified 15 miRNAs that can differentiate normal versus cancer tissues, and a unique 20-miRNA metastasis signature was defined that could significantly predict (p < 0.001) primary hepatocellular carcinoma tissues with venous metastasis from metastasis-free solitary tumors (16Budhu A. Jia H.L. Forgues M. Liu C.G. Goldstein D. Lam A. Zanetti K.A. Ye Q.H. Qin L.X. Croce C.M. Tang Z.Y. Wang X.W. Hepatology. 2008; 47: 897-907Crossref PubMed Scopus (621) Google Scholar).Cancer metastasis is a highly complex process that involves alterations in growth, dissemination, invasion, and survival, which leads to subsequent attachment, angiogenesis, and growth of new cancer cell colonies (17Nguyen D.X. Massagué J. Nat. Rev. Genet. 2007; 8: 341-352Crossref PubMed Scopus (631) Google Scholar). miRNAs have profound positive and negative effects on cancer metastasis (18Liang Z. Wu H. Reddy S. Zhu A. Wang S. Blevins D. Yoon Y. Zhang Y. Shim H. Biochem. Biophys. Res. Commun. 2007; 363: 542-546Crossref PubMed Scopus (119) Google Scholar, 19Ma L. Teruya-Feldstein J. Weinberg R.A. Nature. 2007; 449: 682-688Crossref PubMed Scopus (2197) Google Scholar). Overexpression of an artificial miRNA that targeted the CXCR4 gene in the human breast cancer cell line, MDA-MB-231, decreases cell migration and invasion (18Liang Z. Wu H. Reddy S. Zhu A. Wang S. Blevins D. Yoon Y. Zhang Y. Shim H. Biochem. Biophys. Res. Commun. 2007; 363: 542-546Crossref PubMed Scopus (119) Google Scholar). miR-10b is highly expressed in human and mouse metastatic breast cancer cell lines and positively regulates cell migration and metastasis (19Ma L. Teruya-Feldstein J. Weinberg R.A. Nature. 2007; 449: 682-688Crossref PubMed Scopus (2197) Google Scholar).Importantly, miRNA research should seek the real target genes experimentally rather than computationally and provide evidence for their involvement in vital signaling pathways or cell processes, such as development, differentiation, and apoptosis. Hence, understanding miRNA function and target genes is an active area of research. miR-17–5p represses the AIB1 (amplified in breast cancer 1) gene as well as the estrogen receptor target genes, including cyclin D1 and E2F1 target gene CDC2 (20Hossain A. Kuo M.T. Saunders G.F. Mol. Cell Biol. 2006; 26: 8191-8201Crossref PubMed Scopus (447) Google Scholar). miR-21 down-regulates the tumor suppressor gene TPM1 (tropomyosin 1), in MCF7 cells (21Zhu 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) and tumor suppressor PDCD4 genes in colorectal cancer cell lines (22Frankel L.B. Christoffersen N.R. Jacobsen A. Lindow M. Krogh A. Lund A.H. J. Biol. Chem. 2008; 283: 1026-1033Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar). Overexpression of miR-200c can suppress transcription factor 8 and increase the expression of E-cadherin, a key protein that regulates epithelial-mesenchymal transition (23Hurteau G.J. Carlson J.A. Spivack S.D. Brock G.J. Cancer Res. 2007; 67: 7972-7976Crossref PubMed Scopus (366) Google Scholar, 24Gregory P.A. Bert A.G. Paterson E.L. Barry S.C. Tsykin A. Farshid G. Vadas M.A. Khew-Goodall Y. Goodall G.J. Nat. Cell Biol. 2008; 10: 593-601Crossref PubMed Scopus (3116) Google Scholar). Repression of estrogen receptor-α by miR-206 occurs in human breast cancer cell lines (25Adams B.D. Furneaux H. White B.A. Mol. Endocrinol. 2007; 21: 1132-1147Crossref PubMed Scopus (412) Google Scholar), whereas miR-125a and miR-125b overexpression could suppress ERBB2 and ERBB3 expression (26Scott G.K. Goga A. Bhaumik D. Berger C.E. Sullivan C.S. Benz C.C. J. Biol. Chem. 2007; 282: 1479-1486Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). These data clearly indicate that miRNAs play important roles in several biological processes.ST14 is an epithelium-derived membrane serine protease. ST14 forms a complex with a serine-protease inhibitor, HAI-1 (27Vogel L.K. Saebø M. Skjelbred C.F. Abell K. Pedersen E.D. Vogel U. Kure E.H. BMC Cancer. 2006; 6: 176Crossref PubMed Scopus (61) Google Scholar). The expression of ST14 has been associated with several tumors, including breast, colon, prostate, and ovary (28Darragh M.R. Bhatt A.S. Craik C.S. Front. Biosci. 2008; 13: 528-539Crossref PubMed Scopus (35) Google Scholar). Prostate tumor cells, LNCap, have extremely low ST14 activity although expression levels of ST14 are high in these cells. Interestingly, stimulation of ST14/matriptase activity increased the ability of LNCap cells to invade (29Tsui K.H. Chang P.L. Feng T.H. Chung L.C. Sung H.C. Juang H.H. Anticancer Res. 2008; 28: 1993-1999PubMed Google Scholar). In contrast, ST14 levels are down-regulated in SK-Hep1 human hepatoma cells, suggesting a tumor-suppressive function of ST14 (30Hwang E.S. Kim G.H. Exp. Biol. Med. 2009; 234: 105-111Crossref Scopus (16) Google Scholar). Matriptase RNA levels are low in colorectal carcinoma tissues compared with normal tissues from healthy individuals as well as with their adjacent normal tissue (27Vogel L.K. Saebø M. Skjelbred C.F. Abell K. Pedersen E.D. Vogel U. Kure E.H. BMC Cancer. 2006; 6: 176Crossref PubMed Scopus (61) Google Scholar). These data indicate that ST14/matriptase plays an important role in several types of cancers. Relatively little is known about mechanisms regulating ST14 in cancer.Here, we profiled a highly invasive human breast cancer cell line, MDA-MB-231 subline 4175 (henceforth referred to as 4175), and a noninvasive human breast epithelial cell line, MCF10A, by miRNA microarrays and found that expression levels of miR-27b were significantly different. We validated these differences by multiple approaches and used several bioinformatics tools to predict the target genes for these miRNAs. The target for miR-27b is ST14; both miR-27b and ST14 show opposing expression patterns in MCF10A and 4175 cells. Further, the regulatory roles of miR-27b on ST14 in human breast cancer cells were determined using luciferase reporter assays. Our study is the first to reveal that miR-27b negatively regulates ST14 through a specific regulatory element located in its 3′-UTR region. We also examined the expression levels of miR-27b in a panel of 26 human breast cancer and 29 normal breast samples and found higher expression of miR-27b in cancer samples, indicating the importance of miR-27b as a tumor marker. Also, pre-miR-27b enhances cell growth of nonmalignant cells, whereas ST14 inhibits cell growth of highly invasive cells. Also, we demonstrate that ST14 plays an important role in cell cycle during G1 to S progression. Cells expressing ST14 exhibited a defect in S phase entry with down-regulation of cyclin E and CDK2, indicating that ST14 arrests cells at the G1/S boundary. Surprisingly, the effect of ST14 on cell growth is not related to miR-27b regulation, suggesting miR-27b-dependent and -independent functions of ST14 exist. Furthermore, anti-miR-27b blocks cell invasion, and a precursor of miR-27b (pre-miR-27b) stimulates cell invasion. In addition, ST14 significantly reduces cell invasion. Our results reveal that miR-27b may function as an oncogene, whereas ST14 may function as a tumor suppressor in breast cancer cells. So far, it has not been investigated whether ST14 is being regulated by miRNAs. This is the first report demonstrating that miR-27b via a specific target at nucleotides 115–122 of the 3′-UTR is negatively regulating ST14.DISCUSSIONSeveral miRNAs function as tumor suppressors or oncogenes and regulate target gene expression at a posttranscriptional level (7Zhang B. Pan X. Cobb G.P. Anderson T.A. Dev. Biol. 2007; 302: 1-12Crossref PubMed Scopus (2136) Google Scholar). miRNAs perform important roles in human breast cancer cell growth (13Iorio M.V. Ferracin M. Liu C.G. Veronese A. Spizzo R. Sabbioni S. Magri E. Pedriali M. Fabbri M. Campiglio M. Ménard 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 (3437) Google Scholar, 21Zhu 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) and breast cancer metastasis (19Ma L. Teruya-Feldstein J. Weinberg R.A. Nature. 2007; 449: 682-688Crossref PubMed Scopus (2197) Google Scholar). However, a comparison of a highly metastatic (lung-specific) human breast cancer cell line, 4175, and the human breast epithelial cell line, MCF10A, at the mature miRNA level has not been reported. In this study, we profiled the expression patterns of 471 mature miRNAs in these two cell lines and found differential expression of 22 miRNAs (p < 0.05). The expression patterns of miR-27b, miR-99a (data not shown), and miR-205a (data not shown) were validated by real time PCR and Northern blotting, showing that our microarray is reliable.We found that miR-21 and miR-205 were expressed at lower levels in 4175 cells than in MCF10A cells. miR-205 is also down-regulated in prostate hormone-refractory cancerous tissues compared with benign prostatic hyperplasia (12Porkka K.P. Pfeiffer M.J. Waltering K.K. Vessella R.L. Tammela T.L. Visakorpi T. Cancer Res. 2007; 67: 6130-6135Crossref PubMed Scopus (785) Google Scholar). In contrast, both miR-21 and miR-205 act as oncogenes and are up-regulated in cancerous tissues in lungs (9Yanaihara N. Caplen N. Bowman E. Seike M. Kumamoto K. Yi M. Stephens R.M. Okamoto A. Yokota J. Tanaka T. Calin G.A. Liu C.G. Croce C.M. Harris C.C. Cancer Cell. 2006; 9: 189-198Abstract Full Text Full Text PDF PubMed Scopus (2675) Google Scholar), in breast (13Iorio M.V. Ferracin M. Liu C.G. Veronese A. Spizzo R. Sabbioni S. Magri E. Pedriali M. Fabbri M. Campiglio M. Ménard 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 (3437) Google Scholar), in tonsils (15Tran N. McLean T. Zhang X. Zhao C.J. Thomson J.M. O'Brien C. Rose B. Biochem. Biophys. Res. Commun. 2007; 358: 12-17Crossref PubMed Scopus (226) Google Scholar), in ovaries (10Iorio M.V. Visone R. Di Leva G. Donati V. Petrocca F. Casalini P. Taccioli C. Volinia S. Liu C.G. Alder H. Calin G.A. Menard S. Croce C.M. Cancer Res. 2007; 67: 8699-8707Crossref PubMed Scopus (1287) Google Scholar), and in head and neck cancer cell lines (15Tran N. McLean T. Zhang X. Zhao C.J. Thomson J.M. O'Brien C. Rose B. Biochem. Biophys. Res. Commun. 2007; 358: 12-17Crossref PubMed Scopus (226) Google Scholar). Recently, a study by Gregory et al. indicated that miR-200 regulates the epithelial to mesenchymal transition by targeting Zeb1 and Sip1 proteins, thereby modulating tumor metastases (24Gregory P.A. Bert A.G. Paterson E.L. Barry S.C. Tsykin A. Farshid G. Vadas M.A. Khew-Goodall Y. Goodall G.J. Nat. Cell Biol. 2008; 10: 593-601Crossref PubMed Scopus (3116) Google Scholar). miR-200 and miR-205 also may help establish epithelial cell lineages during embryonic development. Thus, the differential expression of miRNAs is not surprising, because miRNAs can be expressed in tissue-specific, cell line-specific, or cancer type-specific manners (8Jiang J. Lee E.J. Gusev Y. Schmittgen T.D. Nucleic Acids Res. 2005; 33: 5394-5403Crossref PubMed Scopus (447) Google Scholar).miRNAs are involved in many signaling pathways by regulating target genes. Although a bioinformatics analysis comparing 3′-UTRs and miRNAs can reveal multiple target genes, these targets must be confirmed experimentally. We used three online tools, TargetScan, PicTar, and Miranda, to predict the targets for miR-27b and identified ST14 and others. Through the luciferase assay, we demonstrated that at least one effective binding site is present in the ST14 3′-UTR; consistently, we observed that miR-27b is inversely correlated with ST14 in several breast cancer cell lines and tissues. We provided functional evidence of the possible role of ST14 in breast cancer by showing that forced expression of ST14 is able to reduce cell growth potential.ST14, also named TADG-15 and MT-SP1, is a transmembrane serine protease involved in ECM degradation and may be involved in cancer migration, invasion, or metastasis (28Darragh M.R. Bhatt A.S. Craik C.S. Front. Biosci. 2008; 13: 528-539Crossref PubMed Scopus (35) Google Scholar). ST14 is overexpressed in ovarian tumors, but patients with ST14-positive tumors had substantially longer survival, suggesting that ST14 might be a prognostic marker for ovarian carcinoma (38Tanimoto H. Shigemasa K. Tian X. Gu L. Beard J.B. Sawasaki T. O'Brien T.J. Br. J. Cancer. 2005; 92: 278-283Crossref PubMed Scopus (51) Google Scholar). Our results show that the 3′-UTR of ST14 carries a putative binding site for miR-27b, and these two molecules show inverted expression patterns, indicating that ST14 is a target of miR-27b in breast cancer cells. We further evaluated miR-27b expression in matched sets of normal tissues and primary breast tumors, and again miR-27b expression was high in tumors. miR-27b expression levels are significantly higher in IDC tumors compared with normal samples (p < 0.01). Our ST14 expression data in breast cancer patients and normals indicate that ST14 expression is elevated in normals compared with cancer tissues. Also, our data show that expression levels of miR-27b and ST14 have an inverse relationship in all tissues. Furthermore, our data revealed that miR-27b promotes cell proliferation, cell migration, and cell invasion, characteristics of oncogenes. This is the first study to describe the potential oncogenic role of miR-27b in breast tumorigenesis and to show loss of expression of ST14 in breast cancer.Our results indicate that miR-27b may function as an oncogene, whereas another study suggested it might function as a tumor suppressor despite its expression in MCF7 cells (41Tsuchiya Y. Nakajima M. Takagi S. Taniya T. Yokoi T. Cancer Res. 2006; 66: 9090-9098Crossref PubMed Scopus (345) Google Scholar). Our study shows that miR-27b is overexpressed in highly invasive breast cancer cells, with expression increasing in normal (MCF10A) to moderately invasive (MCF7) to highly metastatic (MDA-MB-231) to highly metastatic exclusively to the lung (4175). miR-27a, a close homolog of miR-27b, also functions as an oncogene (42Mertens-Talcott S.U. Chintharlapalli S. Li X. Safe S. Cancer Res. 2007; 67: 11001-11011Crossref PubMed Scopus (411) Google Scholar). Suppression of miR-27a in MDA-MB-231 cells decreased the expression of angiogenic genes and the percentage of MDA-MB-231 cells in S phase of the cell cycle (42Mertens-Talcott S.U. Chintharlapalli S. Li X. Safe S. Cancer Res. 2007; 67: 11001-11011Crossref PubMed Scopus (411) Google Scholar). Decreased ST14 expression could result from the increased expression of miR-27b. In contrast to our findings, as discussed above, a Japanese study reported that miR-27b functions as a tumor suppressor of breast cancer in Japanese patients (41Tsuchiya Y. Nakajima M. Takagi S. Taniya T. Yokoi T. Cancer Res. 2006; 66: 9090-9098Crossref PubMed Scopus (345) Google Scholar). These differences may occur because patients originated from two different continents (Asia and North America) or differed in their tumor status, estrogen receptor/prostaglandin receptor status, or histologic tumor grade. The patients in our study were estrogen receptor+/− or prostaglandin receptor+/−, and there was no association between levels of these receptors and miR-27b levels. In summary, miR-27b decreases ST14 expression in cancer cells. This is the first study to demonstrate that ST14 is negatively regulated by miR-27b post-transcriptionally via a target site in the 3′-UTR. This is also the first study to demonstrate that miR-27b stimulates invasion in breast cancer cells.ST14/TADG15 expression is high in stage I ovarian tumors and lower in stage II/III/IV tumors (38Tanimoto H. Shigemasa K. Tian X. Gu L. Beard J.B. Sawasaki T. O'Brien T.J. Br. J. Cancer. 2005; 92: 278-283Crossref PubMed Scopus (51) Google Scholar). ST14 was identified through a subtractive hybridization analysis by Zhang et al. (43Zhang Y. Cai X. Schlegelberger B. Zheng S. Cytogenet. Cell Genet. 1998; 83: 56-57Crossref PubMed Google Scholar). Also, MT-SP1 was identified in the human prostate cancer cell line, PC3, by Takeuchi et al. (44Takeuchi T. Harris J.L. Huang W. Yan K.W. Coughlin S.R. Craik C.S. J. Biol. Chem. 2000; 275: 26333-26342Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). ST14/TADG15 substrates include hepatocyte growth factor, scattering factor, PAR2 (protease-activated receptor), and urokinase plasminogen activator, which affect cell proliferation, cell motility, and cell invasion (44Takeuchi T. Harris J.L. Huang W. Yan K.W. Coughlin S.R. Craik C.S. J. Biol. Chem. 2000; 275: 26333-26342Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 45Parr C. Sanders A.J. Davies G. Martin T. Lane J. Mason M.D. Mansel R.E. Jiang W.G. Clin. Cancer Res. 2007; 13: 3568-3576Crossref PubMed Scopus (44) Google Scholar, 46Parr C. Jiang W.G. Int. J. Cancer. 2006; 119: 1176-1183Crossref PubMed Scopus (75) Google Scholar). ST14 may serve as a marker for early detection of ovarian cancer. Our data indicate that ST14 reduces cell growth as well as cell invasion and cell migration, suggesting that ST14 may function as a tumor suppressor. However, TADG15 is overexpressed in 100% of primary squamous cervical tumors and 40% of cervical adenocarcinoma cell lines but not normal cervical keratinocyte control cell lines (47Santin A.D. Cane S. Bellone S. Bignotti E. Palmieri M. De Las Casas L.E. Anfossi S. Roman J.J. O'Brien T. Pecorelli S. Cancer. 2003; 98: 1898-1904Crossref PubMed Scopus (52) Google Scholar). Overexpression of ST14 enhances cell adhesion in colorectal cancer cells (48Sun L.F. Zheng S. Shi Y. Fang X.M. Ge W.T. Ding K.F. Zhonghua Yi Xue Za Zhi. 2004; 84: 843-848PubMed Google Scholar). Thus, ST14 has different functions in different cancer cell types, and it will be very interesting to study the in vitro and in vivo functions of ST14 in different cancers.In the cell cycle of mammalian cells, cell division and growth are tightly controlled by a series of positive and negative regulators that act at several points throughout the cell cycle. Our data indicate that the inhibitory effect of ST14 is exerted at some point in the G1 phase. The inhibition of DNA synthesis by ST14 is associated with a reduction in the proportion of cells in the S phase and more number of cells in the G1 phase. Hence, we specifically focused on proteins that regulate the cell cycle. CDKs play an important role and promote G1/S transition by phosphorylation of the pRb protein. CDK inhibitors, such as p27Kip1 and p21Cip1 inhibit their kinase activity. ST14 down-regulates CDK2 and cyclin E expression, but not CDK4 and cyclin D1 expression. In the current study, the expression levels of p27Kip1 distinctly increased after treatment with ST14. Accordingly, current results suggest that the increase in the protein levels of p27Kip1 was accompanied by a decrease in the expression level of CDKs in the G1 phase, eventually leading to a cell cycle arrest of the G1 phase in the 4175 cells expressing ST14. However, the detailed mechanism still needs to be studied further. Furthermore, to our surprise, miR-27b failed to rescue the inhibitory effect of ST14 on cell proliferation, suggesting that the effects may be independent of connection between ST14 and miR-27b. Thus, it will be of great interest for us to understand the mechanisms of miR-27b-dependent and independent functions of ST14 in breast cancer in the near future.Our findings are of high importance to the understanding of breast tumorigenesis, since they directly link ST14 to the regulation of cyclin-CDK2 complexes. Cyclin E plays an important role in regulation of G1/S phase transition and in tumorigenesis. Our studies provide evidence for an additional level of control in regulation of the G1/S phase transition. Cyclin D, similar to cyclin E, can enhance G1 transition; however, cyclin D1 is not regulated by ST14. The cyclin D1 and E phosphorylate and inactivate pRb protein, a well known tumor suppressor. pRb phosphorylation plays a pivotal role in controlling cellular proliferation by regulating the G1/S transition of the cell cycle. During continuous proliferation of cells, pRb is phosphorylated by the activity of G1 phase CDKs, such as CDK4/6 and CDK2, thereby liberating the factors that control the S phase entrance (40Deshpande A. Sicinski P. Hinds P.W. Oncogene. 2005; 24: 2909-2915Crossref PubMed Scopus (344) Google Scholar, 49Buttitta L.A. Edgar B.A. Curr. Opin. Cell Biol. 2007; 19: 697-704Crossref PubMed Scopus (142) Google Scholar, 50DeGregori J. Mol. Cell Biol. 2006; 26: 1165-1169Crossref PubMed Scopus (12) Google Scholar). Thus, ST14 may affect factors that regulate the S phase entry, which will be the subject of our future investigation. The current studies are the first to demonstrate that cyclin E-CDK2 complexes are controlled by ST14. In summary, our study suggests that ST14 may function as a tumor suppressor and miR-27b induces invasion and cell growth of breast cancer cells. MicroRNAs (miRNAs) 2The abbreviations used are: miRNAmicroRNAsUTRuntranslated regionRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenaseQPCRreal time quantitative PCRX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideIDCinvasive ductal carcinomaCDKcyclin-dependent kinasepRbretinoblastoma protein. 2The abbreviations used are: miRNAmicroRNAsUTRuntranslated
Referência(s)