title: Novel MicroRNA Reporter Uncovers Repression of Let-7 by GSK-3β.
abstract: Several members of the let-7 microRNA family are downregulated in ovarian and other cancers. They are thought to act as tumor suppressors by lowering growth-promoting and anti-apoptotic proteins. In order to measure cellular let-7 levels systematically, we have developed a highly sensitive let-7 reporter assay system based on the expression of a chimeric mRNA that contains the luciferase coding region and a 3'-untranslated region (UTR) bearing two let-7-binding sites. In cells expressing the reporter construct, termed pmirGLO-let7, luciferase activity was high when let-7 was absent, while luciferase activity was low when let-7 levels were elevated. The ovarian cancer cell lines BG-1 and UCI-101 were transfected with the let-7 reporter and surveyed with a library of kinase inhibitors in order to identify pathways affecting let-7 activity. Among the inhibitors causing changes in endogenous let-7 abundance, the lowering of glycogen synthase kinase 3 (GSK-3)β function specifically increased let-7 levels and lowered luciferase activity. Similarly, silencing GSK-3β increased both mature and primary-let-7 levels in BG-1 cells, and decreased BG-1 cell survival. Further studies identified p53 as a downstream effector of the GSK-3β-mediated repression of let-7 biosynthesis. Our studies highlight GSK-3β as a novel therapeutic target in ovarian tumorigenesis.
authors.affiliation: Laboratory of Genetics, National Institute on Aging-Intramural Research Program, NIH, Baltimore, Maryland, United States of America.
authors.forename: Rong
authors.initials: R
authors.lastname: Guo
authors.forename: Kotb
authors.initials: K
authors.lastname: Abdelmohsen
authors.forename: Patrice J
authors.initials: PJ
authors.lastname: Morin
authors.forename: Myriam
authors.initials: M
authors.lastname: Gorospe
mesh_terms: D045744:Cell Line, Tumor; D016922:Cellular Senescence; D005260:Female; D016147:Genes, Tumor Suppressor; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D035683:MicroRNAs; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C487931:mirnlet7 microRNA, human; D000071679:Glycogen Synthase Kinase 3 beta