MicroRNA-19 (miR-19) Regulates Tissue Factor Expression in Breast Cancer Cells*

Tissue factor has been recognized as a regulator of tumor angiogenesis and metastasis. The tissue factor gene is selectively expressed in highly invasive breast cancer cells, and the mechanisms regulating tissue factor expression in these cells remain unclear. This study demonstrates that microRNA-19 (miR-19) regulates tissue factor expression in breast cancer cells, providing a molecular basis for the selective expression of the tissue factor gene. Tissue factor protein was barely detectable in MCF-7, T47D, and ZR-75-1 cells (less invasive breast lines) but was expressed at a significantly higher level in MDA-MB-231 and BT-20 cells (invasive breast lines) as assayed by Western blot. The tissue factor gene promoter was activated, and forced expression of tissue factor cDNA was achieved in MCF-7 cells, implying that the 3′-UTR of the tissue factor transcript is responsible for the suppression of tissue factor expression. Bioinformatics analysis predicted microRNA-binding sites for miR-19, miR-20, and miR-106b in the 3′-UTR of the tissue factor transcript. Reporter gene assay using the TF-3′-UTR luciferase reporter construct confirmed that the 3′-UTR negatively regulates gene expression in MCF-7 cells, an effect reversed by deletion of the miR-19-binding site. Application of the miR-19 inhibitor induces endogenous tissue factor expression in MCF-7 cells, and overexpression of miR-19 down-regulates tissue factor expression in MDA-MB-231 cells. RT-PCR analysis using cDNA made from Ago2-immunoprecipitated RNA samples confirmed that Ago2 binds preferentially to tissue factor 3′-UTR in MCF-7 cells, as compared with MDA-MB-231 cells, consistent with the observation that miR-19 levels are higher in MCF-7 cells.

[1]  N. Mackman.  Regulation of the Tissue Factor Gene , 1997, Thrombosis and Haemostasis.

[2]  E. Furth,et al.  Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster , 2006, Nature Genetics.

[3]  J. Kitzman,et al.  Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. , 2007, RNA.

[4]  J. Rak,et al.  The hemostatic system and angiogenesis in malignancy. , 2001, Neoplasia.

[5]  M. Reed,et al.  Tissue factor, angiogenesis and tumour progression , 2008, Breast Cancer Research.

[6]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[7]  E. Izaurralde,et al.  Getting to the Root of miRNA-Mediated Gene Silencing , 2008, Cell.

[8]  Jie Zhou,et al.  microRNA 17/20 inhibits cellular invasion and tumor metastasis in breast cancer by heterotypic signaling , 2010, Proceedings of the National Academy of Sciences.

[9]  N. Sangster-Guity,et al.  Molecular profiling of embryonal carcinoma cells following retinoic acid or histone deacetylase inhibitor treatment , 2004, Cancer biology & therapy.

[10]  S. Coughlin,et al.  Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Provost,et al.  Protein components of the microRNA pathway and human diseases. , 2009, Methods in molecular biology.

[12]  D. Tong,et al.  Association of in vitro invasiveness and gene expression of estrogen receptor, progesterone receptor, pS2 and plasminogen activator inhibitor‐1 in human breast cancer cell lines , 1999, Breast Cancer Research and Treatment.

[13]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

[14]  S. Linder,et al.  Activation of tissue‐factor gene expression in breast carcinoma cells by stimulation of the RAF‐ERK signaling pathway , 1998, Molecular carcinogenesis.

[15]  W. Ruf,et al.  Tissue factor and PAR2 signaling in the tumor microenvironment. , 2009, Arteriosclerosis, thrombosis, and vascular biology.

[16]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[17]  M. Ihnat,et al.  Docosahexaenoic Acid Inhibits Superoxide Dismutase 1 Gene Transcription in Human Cancer Cells: The Involvement of Peroxisome Proliferator-Activated Receptor α and Hypoxia-Inducible Factor-2α Signaling , 2009, Molecular Pharmacology.

[18]  J. Mendell.  miRiad Roles for the miR-17-92 Cluster in Development and Disease , 2008, Cell.

[19]  J. Rak,et al.  Oncogenes as regulators of tissue factor expression in cancer: implications for tumor angiogenesis and anti-cancer therapy. , 2004, Seminars in thrombosis and hemostasis.

[20]  W. Ding,et al.  Differential sensitivity of cancer cells to docosahexaenoic acid-induced cytotoxicity: the potential importance of down-regulation of superoxide dismutase 1 expression. , 2004, Molecular cancer therapeutics.

[21]  M. Hendrix,et al.  Differentially expressed genes associated with the metastatic phenotype in breast cancer , 1999, Breast Cancer Research and Treatment.

[22]  Lin Zhang,et al.  The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis , 2008, Nature Cell Biology.

[23]  N. Tsopanoglou,et al.  Inhibition of angiogenesis by small-molecule antagonists of protease-activated receptor-1. , 2007, Seminars in thrombosis and hemostasis.

[24]  Ming-Ta Hsu,et al.  Genome-wide mapping and characterization of hypomethylated sites in human tissues and breast cancer cell lines. , 2008, Genome research.

[25]  M. Volm,et al.  Tissue‐factor expression in human non‐small‐cell lung carcinoma measured by immunohistochemistry: Correlation between tissue factor and angiogenesis , 1998, International journal of cancer.

[26]  J. Steitz,et al.  miRNPs: versatile regulators of gene expression in vertebrate cells. , 2009, Biochemical Society transactions.

[27]  R. Weinberg,et al.  Tumour invasion and metastasis initiated by microRNA-10b in breast cancer , 2007, Nature.

[28]  S. Nakamura,et al.  Tissue factor expression in breast cancer tissues: its correlation with prognosis and plasma concentration , 2000, British Journal of Cancer.

[29]  Y. Yatabe,et al.  A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. , 2005, Cancer research.

[30]  Massimo Negrini,et al.  Breast cancer metastasis: a microRNA story , 2008, Breast Cancer Research.

[31]  Stefanie Dimmeler,et al.  Targeting microRNA expression to regulate angiogenesis. , 2008, Trends in pharmacological sciences.

[32]  C. Croce,et al.  MicroRNA-cancer connection: the beginning of a new tale. , 2006, Cancer research.

[33]  T. Miyata,et al.  Regulation of human tissue factor expression by mRNA turnover. , 1993, The Journal of biological chemistry.

[34]  N. Mackman,et al.  Tissue factor mRNA in THP-1 monocytic cells is regulated at both transcriptional and posttranscriptional levels in response to lipopolysaccharide , 1991, Molecular and cellular biology.

[35]  C. Croce,et al.  A microRNA expression signature of human solid tumors defines cancer gene targets , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[36]  O. Matsubara,et al.  Expression of tissue factor in non-small-cell lung cancers and its relationship to metastasis , 1999, British Journal of Cancer.

[37]  D. Medina,et al.  Re-evaluation of mammary stem cell biology based on in vivo transplantation , 2008, Breast Cancer Research.

[38]  G. Hair,et al.  In situ detection of tissue factor in vascular endothelial cells: Correlation with the malignant phenotype of human breast disease , 1996, Nature Medicine.

[39]  H. Prydz,et al.  Coagulation Factors VIIa and Xa Induce Cell Signaling Leading to Up-regulation of the egr-1 Gene* , 1999, The Journal of Biological Chemistry.

[40]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[41]  Kathryn A. O’Donnell,et al.  c-Myc-regulated microRNAs modulate E2F1 expression , 2005, Nature.

[42]  Terry Hyslop,et al.  A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation , 2008, The Journal of cell biology.

[43]  K. Picha,et al.  CNTO 859, a humanized anti‐tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models , 2007, International journal of cancer.

[44]  Xiaofeng Jiang,et al.  Formation of tissue factor–factor VIIa–factor Xa complex promotes cellular signaling and migration of human breast cancer cells , 2004, Journal of thrombosis and haemostasis : JTH.

[45]  Yuriy Gusev,et al.  Computational methods for analysis of cellular functions and pathways collectively targeted by differentially expressed microRNA. , 2008, Methods.