title: Regulation of the let-7a-3 promoter by NF-κB.
abstract: Changes in microRNA expression have been linked to a wide array of pathological states. However, little is known about the regulation of microRNA expression. The let-7 microRNA is a tumor suppressor that inhibits cellular proliferation and promotes differentiation, and is frequently lost in tumors. We investigated the transcriptional regulation of two let-7 family members, let-7a-3 and let-7b, which form a microRNA cluster and are located 864 bp apart on chromosome 22q13.31. Previous reports present conflicting data on the role of the NF-κB transcription factor in regulating let-7. We cloned three fragments upstream of the let-7a-3/let-7b miRNA genomic region into a plasmid containing a luciferase reporter gene. Ectopic expression of subunits of NF-κB (p50 or p65/RelA) significantly increased luciferase activity in HeLa, 293, 293T and 3T3 cells, indicating that the let-7a-3/let-7b promoter is highly responsive to NF-κB. Mutation of a putative NF-κB binding site at bp -833 reduced basal promoter activity and decreased promoter activity in the presence of p50 or p65 overexpression. Mutation of a second putative binding site, at bp -947 also decreased promoter activity basally and in response to p65 induction, indicating that both sites contribute to NF-κB responsiveness. While the levels of the endogenous primary let-7a and let-7b transcript were induced in response to NF-κB overexpression in 293T cells, the levels of fully processed, mature let-7a and let-7b miRNAs did not increase. Instead, levels of Lin-28B, a protein that blocks let-7 maturation, were induced by NF-κB. Increased Lin-28B levels could contribute to the lack of an increase in mature let-7a and let-7b. Our results suggest that the final biological outcome of NF-κB activation on let-7 expression may vary depending upon the cellular context. We discuss our results in the context of NF-κB activity in repressing self-renewal and promoting differentiation.
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.
authors.forename: David J
authors.initials: DJ
authors.lastname: Wang
authors.forename: Aster
authors.initials: A
authors.lastname: Legesse-Miller
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Johnson
authors.forename: Hilary A
authors.initials: HA
authors.lastname: Coller
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000502:physiology*; D016328:NF-kappa B / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins
chemical_list: C000715187:Lin28A protein, human; D035683:MicroRNAs; D016328:NF-kappa B; D016601:RNA-Binding Proteins; C487931:mirnlet7 microRNA, human; C542687:mirnlet7 microRNA, mouse