MicroRNA miR-21 Regulates the Metastatic Behavior of B16 Melanoma Cells*

Background: miRNAs are involved in many critical biological processes. Results: miR-21 induction is STAT3-dependent, and miR-21 knockdown inhibited melanoma cell proliferation and migration and enhanced apoptosis. Although B16 cells produced large lung metastases in mice, miR-21 knockdown cells only formed small lung lesions. Conclusion: miR-21 regulates the metastatic behavior of melanoma. Significance: miR-21 is identified as a potential drug target in melanoma. MicroRNA-21 (miR-21) is overexpressed in many human tumors and has been linked to various cellular processes altered in cancer. miR-21 is also up-regulated by a number of inflammatory agents, including IFN, which is of particular interest considering the close relationship between inflammation and cancer. Because miR-21 appears to be overexpressed in human melanoma, we examined the role of miR-21 in cancer development and metastasis in B16 mouse melanoma cells. We found that miR-21 is a member of an IFN-induced miRNA subset that requires STAT3 activation. To characterize the role of miR-21 in melanoma behavior, we transduced B16 cells with lentivirus encoding a miR-21 antagomir and isolated miR-21 knockdown B16 cells. miR-21 knockdown or IFN treatment alone inhibited B16 cell proliferation and migration in vitro, and in combination they had an enhanced effect. Moreover, miR-21 knockdown sensitized B16 cells to IFN-induced apoptosis. In B16 cells miR-21 targeted tumor suppressor (PTEN and PDCD4) and antiproliferative (BTG2) proteins. To characterize the role of miR-21 in vivo, empty vector- and antagomiR-21-transduced B16 melanoma cells were injected via tail vein into syngeneic C57BL/6 mice. Although empty vector-transduced B16 cells produced large lung metastases, miR-21 knockdown cells only formed small lung lesions. Importantly, miR-21 knockdown tumor-bearing mice exhibited prolonged survival compared with empty vector tumor-bearing mice. Thus, miR-21 regulates the metastatic behavior of B16 melanoma cells by promoting cell proliferation, survival, and migration/invasion as well as by suppressing IFN action, providing important new insights into the role of miR-21 in melanoma.

[1]  Danish Sayed,et al.  MicroRNA-21 targets Sprouty2 and promotes cellular outgrowths. , 2008, Molecular biology of the cell.

[2]  Anna M. Krichevsky,et al.  miR-21: a small multi-faceted RNA , 2008, Journal of cellular and molecular medicine.

[3]  J. Kirkwood,et al.  Interferon-alpha in tumor immunity and immunotherapy. , 2002, Cytokine & growth factor reviews.

[4]  Roy Garcia,et al.  STATs in oncogenesis , 2000, Oncogene.

[5]  L. Pfeffer,et al.  The role of constitutively activated STAT3 in B16 melanoma cells. , 2010, International journal of interferon, cytokine and mediator research.

[6]  F. Slack,et al.  OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma , 2010, Nature.

[7]  L. Pfeffer,et al.  IFNα/β promotes cell survival by activating NF-κB , 2000 .

[8]  G. Poste,et al.  Comparison of the metastatic properties of B16 melanoma clones isolated from cultured cell lines, subcutaneous tumors, and individual lung metastases. , 1982, Cancer research.

[9]  C. Croce,et al.  MicroRNA signatures in human cancers , 2006, Nature Reviews Cancer.

[10]  L. Pfeffer,et al.  Interferon α/β Promotes Cell Survival by Activating Nuclear Factor κB through Phosphatidylinositol 3-Kinase and Akt* , 2001, The Journal of Biological Chemistry.

[11]  Munish Kumar,et al.  Loss of the miR-21 allele elevates the expression of its target genes and reduces tumorigenesis , 2011, Proceedings of the National Academy of Sciences.

[12]  E. Olson,et al.  Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21. , 2010, Cancer cell.

[13]  S Pestka,et al.  Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons. , 1998, Cancer research.

[14]  K. Kosik,et al.  MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. , 2005, Cancer research.

[15]  S. Binkley.  A timekeeping enzyme in the pineal gland. , 1979, Scientific American.

[16]  L. Pfeffer,et al.  Cancer esearch apeutics , Targets , and Chemical Biology Induces miR-21 through a Signal Transducer and ivator of Transcription 3 – Dependent Pathway as a R pressive Negative Feedback on IFN-Induced Apoptosis , 2010 .

[17]  K. Ghoshal,et al.  MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. , 2007, Gastroenterology.

[18]  L. Pfeffer,et al.  Interferon Induces NF-κB-inducing Kinase/Tumor Necrosis Factor Receptor-associated Factor-dependent NF-κB Activation to Promote Cell Survival* , 2005, Journal of Biological Chemistry.

[19]  G. Nicolson,et al.  Specificity of arrest, survival, and growth of selected metastatic variant cell lines. , 1978, Cancer research.

[20]  Jörg Hackermüller,et al.  Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. , 2007, Blood.

[21]  Shuomin Zhu,et al.  MicroRNA-21 targets tumor suppressor genes in invasion and metastasis , 2008, Cell Research.

[22]  Silvia Veneroni,et al.  miR-21: an oncomir on strike in prostate cancer , 2010, Molecular Cancer.

[23]  Shuomin Zhu,et al.  miR-21-mediated tumor growth , 2007, Oncogene.

[24]  J. Darnell,et al.  Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. , 1994, Science.

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

[26]  L. Pfeffer,et al.  STAT3 as an adapter to couple phosphatidylinositol 3-kinase to the IFNAR1 chain of the type I interferon receptor. , 1997, Science.

[27]  Zhenghe Wang,et al.  microRNA-21 negatively regulates Cdc25A and cell cycle progression in colon cancer cells. , 2009, Cancer research.

[28]  H. Allgayer,et al.  MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer , 2008, Oncogene.

[29]  Stefano Volinia,et al.  Interferon modulation of cellular microRNAs as an antiviral mechanism , 2007, Nature.

[30]  J. Turkson,et al.  STAT proteins: novel molecular targets for cancer drug discovery , 2000, Oncogene.

[31]  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.

[32]  A. Krogh,et al.  Programmed Cell Death 4 (PDCD4) Is an Important Functional Target of the MicroRNA miR-21 in Breast Cancer Cells* , 2008, Journal of Biological Chemistry.