title: Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling.
abstract: A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ~90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a-regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division.
authors.affiliation: Immune Disease Institute, Program in Cellular and Molecular Medicine, Children's Hospital Boston, Boston, Massachusetts, United States of America. lalas@mail.nih.gov
authors.forename: Ashish
authors.initials: A
authors.lastname: Lal
authors.forename: Marshall P
authors.initials: MP
authors.lastname: Thomas
authors.forename: Gabriel
authors.initials: G
authors.lastname: Altschuler
authors.forename: Francisco
authors.initials: F
authors.lastname: Navarro
authors.forename: Elizabeth
authors.initials: E
authors.lastname: O'Day
authors.forename: Xiao Ling
authors.initials: XL
authors.lastname: Li
authors.forename: Carla
authors.initials: C
authors.lastname: Concepcion
authors.forename: Yoon-Chi
authors.initials: YC
authors.lastname: Han
authors.forename: Jerome
authors.initials: J
authors.lastname: Thiery
authors.forename: Danielle K
authors.initials: DK
authors.lastname: Rajani
authors.forename: Aaron
authors.initials: A
authors.lastname: Deutsch
authors.forename: Oliver
authors.initials: O
authors.lastname: Hofmann
authors.forename: Andrea
authors.initials: A
authors.lastname: Ventura
authors.forename: Winston
authors.initials: W
authors.lastname: Hide
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D049109:Cell Proliferation; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks / Q000235:genetics; D016147:Genes, Tumor Suppressor; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D020014:K562 Cells; D020935:MAP Kinase Signaling System; D035683:MicroRNAs / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D051058:Oncogene Protein v-akt / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D036341:Intercellular Signaling Peptides and Proteins; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D051058:Oncogene Protein v-akt
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