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.
Highlights
MicroRNAs that promote cell differentiation, inhibit cell proliferation, or enhance DNA damage or stress-induced cell cycle arrest or death, and whose expression is reduced in some cancers, are candidate tumor suppressor genes [1]
Depending on cellular context [2], ectopic over-expression of miR-34a induces cell cycle arrest [3], senescence [4] or apoptosis [5]. miR-34a is upregulated by p53 in response to DNA damage [6,7,8], but can be transcriptionally activated independently of p53 [9,10]. miR-34a is located on chromosome 1p36, a locus deleted in neuroblastoma, breast, thyroid, and cervical cancer [11,12]
Cell cycle and DNA repair pathways were enriched in genes down-regulated by miR-34a in both K562 and HCT116 cells. These results suggest that miR-34a directly inhibits growth factor signal transduction and cell cycle progression pathways, culminating in reduced expression of genes needed for cell proliferation
Summary
MicroRNAs (miRNAs) that promote cell differentiation, inhibit cell proliferation, or enhance DNA damage or stress-induced cell cycle arrest or death, and whose expression is reduced in some cancers, are candidate tumor suppressor genes [1]. In this study we sought to understand how miR-34a acts as a tumor suppressor by identifying its direct target genes. MiRNA target prediction algorithms typically predict hundreds to thousands of putative miRNA target genes, but most predicted target genes are not bona fide targets and the best algorithms sometimes miss key targets [17,19,20,21]. Analysis of genes whose mRNA or protein expression decreases when a miRNA is overexpressed or increases when it is antagonized identifies genes that may be either direct targets or indirectly regulated [22]. Biochemical methods to PLoS Genetics | www.plosgenetics.org miR-34a Inhibits Growth Factor Signaling
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