title: MiR-21 enhances melanoma invasiveness via inhibition of tissue inhibitor of metalloproteinases 3 expression: in vivo effects of MiR-21 inhibitor.
abstract: Metastatic melanoma is the most aggressive form of this cancer. It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma. Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity. It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma. It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21. Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls. This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39. The proliferation and migration of miR-21 over-expressing cell lines was not affected. Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression. Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice. Intra-tumoral injections of anti-miR-21 produced similar effects. This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.
authors.affiliation: Department of Surgery, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Martin del Campo
authors.affiliation: Department of Surgery, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Latchana
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Kala M
authors.initials: KM
authors.lastname: Levine
authors.affiliation: Department of Surgery, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Valerie P
authors.initials: VP
authors.lastname: Grignol
authors.affiliation: Department of General Pediatrics, Nationwide Children's Hospital, Columbus, Ohio, United States of America.
authors.forename: Ene T
authors.initials: ET
authors.lastname: Fairchild
authors.affiliation: Department of Neurological Surgery, The Ohio State University, Arthur Giangiacomo James Cancer Hospital and Richard Jack Solove Research Institute, Columbus Ohio, United States of America.
authors.forename: Alena Cristina
authors.initials: AC
authors.lastname: Jaime-Ramirez
authors.affiliation: School of Medicine, Wright State University, Dayton, Ohio, United States of America.
authors.forename: Thao-Vi
authors.initials: TV
authors.lastname: Dao
authors.affiliation: School of Medicine, Wright State University, Dayton, Ohio, United States of America.
authors.forename: Volodymyr I
authors.initials: VI
authors.lastname: Karpa
authors.affiliation: Department of Surgery, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Mary
authors.initials: M
authors.lastname: Carson
authors.affiliation: School of Medicine, Northeast Ohio Medical University, Rootstown, Ohio, United States of America.
authors.forename: Akaansha
authors.initials: A
authors.lastname: Ganju
authors.affiliation: School of Medicine, Wright State University, Dayton, Ohio, United States of America.
authors.forename: Anthony N
authors.initials: AN
authors.lastname: Chan
authors.affiliation: Department of Surgery, The Ohio State University, Columbus, Ohio, United States of America; Department of General Pediatrics, Nationwide Children's Hospital, Columbus, Ohio, United States of America; Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Arthur Giangiacomo James Cancer Hospital and Richard Jack Solove Research Institute, Columbus Ohio, United States of America.
authors.forename: William E
authors.initials: WE
authors.lastname: Carson
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000235:genetics* / Q000473:pathology; D034741:RNA, Small Interfering; D012878:Skin Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics* / Q000378:metabolism
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D034741:RNA, Small Interfering; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3