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. 2011 Nov;7(11):e1002363.
doi: 10.1371/journal.pgen.1002363. Epub 2011 Nov 10.

Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling

Ashish Lal  1 , Marshall P Thomas, Gabriel Altschuler, Francisco Navarro, Elizabeth O'Day, Xiao Ling Li, Carla Concepcion, Yoon-Chi Han, Jerome Thiery, Danielle K Rajani, Aaron Deutsch, Oliver Hofmann, Andrea Ventura, Winston Hide, Judy Lieberman
Affiliations

Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling

Ashish Lal et al. PLoS Genet. 2011 Nov.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The Biotin-miRNA pulldown method.
(A) Schematic of the Bi-miRNA pull-down (PD) assay. (B) Activity of 3′-biotinylated miR-34a (Bi-miR-34a) is similar to unbiotinylated miR-34a mimics by dual luciferase assay performed in HeLa cells cotransfected with psiCHECK-2 vector (black) or psi-CHECK-2 bearing a perfectly complementary sequence to miR-34a (psiCHECK-2-AS-miR-34a, white). Transfection with cel-miR-67 is the control (CTL). Luciferase expression was assayed after 24 hr; results are normalized to cells transfected with the luciferase vector and the CTL miRNA. (C) Bi-miR-34a efficiently silences known miR-34a targets CDK4, CDK6 and MYB. K562 cells were transfected with CTL miRNA, miR-34a, Bi-CTL or Bi-miR-34a mimics for 48 hr. Expression was measured by qRT-PCR normalized to GAPDH. The housekeeping genes SDHA and UBC are negative controls. (D) Cytoplasmic lysates from K562 cells were prepared 48 hr after cotransfection with Bi-CTL (black) miRNA or Bi-miR-34a (white) and a plasmid encoding HA-Ago1, HA-Ago2, or empty vector. Enrichment of miR-34a by HA immunoprecipitation was measured by qRT-PCR normalized to U6. Enrichment of Bi-miR-34a in the HA-immunoprecipitates suggests that Bi-miR-34a is incorporated into RISC. (E) Bi-miR-34a pull-downs optimally enrich targets 24 or 48 hr after transfection. K562 cells were transfected in duplicate with Bi-CTL (black) or Bi-miR-34a (white) mimics for the indicated times. Enrichment of known miR-34a targets (CDK4 and CDK6) or control genes (GAPDH and UBC) was assessed by qRT-PCR relative to GAPDH. (F) The streptavidin pull-down enriches for miR-34a target genes in K562 cells transfected with Bi-CTL (black) or Bi-miR-34a (white) mimics. (G) Known miR-24 target mRNAs (H2AX, E2F2 and MYC) are also pulled down with Bi-miR-24 in HepG2 cells reverse transfected 48 hr earlier with Bi-CTL (black) or Bi-miR-24 (white). Enrichment of target mRNAs in (F) and (G) was analyzed by qRT-PCR relative to SDHA. In all panels, data represent mean ± SD of 3 independent experiments. *, p<0.05, #, p<0.01, **, p<0.005, ##, p<0.001.
Figure 2
Figure 2. miR-34a pulls down transcripts of known and novel direct targets of miR-34a.
(A) Overlap of genes enriched ≥1 SD in gene expression microarray analysis of Bi-miR-34a pull-downs from HCT116 and K562 cells. (B) Enrichment of previously described miR-34a target gene mRNAs in Bi-miR-34a pull-downs from HCT116 and K562 cells. Genes enriched ≥1 SD are indicated in bold. Abs = not expressed. (C) Genes down-regulated by ≥20% after miR-34a over-expression. (D) mRNA expression of candidate miR-34a targets identified by Bi-miR-34a pull-down in both HCT116 and K562 cells decreases after miR-34a over-expression. Cumulative distribution plots compare the extent of mRNA reduction of genes enriched to varying degrees in the Bi-miR-34a pull-down with conserved (cons) or all TargetScan (TScan)-predicted targets. Genes whose mRNAs are more highly enriched in the pull-down are progressively more likely to have reduced expression. (E) Pull-down of 11 mRNAs randomly selected from the set of genes enriched ≥2.5-fold by microarray in both HCT116 and K562 Bi-miR-34a pull-downs is confirmed by qRT-PCR relative to SDHA in K562 cells transfected for 24 hr with Bi-CTL (black) or Bi-miR-34a (white). UBC is a negative control and E2F3 is a positive control. Three previously validated miR-34a targets (AXL, CDK4 and FOXP1) in the random list of genes are indicated in bold. (F) miR-34a over-expression significantly decreases the expression of 9 of 11 of the randomly chosen candidate target genes. K562 cells were transfected with CTL miRNA (black) or miR-34a (white) mimics for 72 hr. Expression of random targets was measured by qRT-PCR normalized to GAPDH. Expression of 2 randomly selected genes (ACSM3 and PCYOX1L) and the housekeeping mRNA UBC didn't change significantly. (G) miR-34a targets the 3′UTR of 10 of 11 randomly chosen targets. Luciferase activity was measured 48 hr after HeLa cells were cotransfected with the luciferase reporter psiCHECK2 bearing the 3′UTR of each gene and CTL miRNA or miR-34a mimics. Results obtained after miR-34a transfection were normalized to CTL miRNA. In (E–G), data represent mean ± SD of 3 independent experiments. *, p<0.05, #, p<0.01, **, p<0.005, ##, p<0.001.
Figure 3
Figure 3. Genes in the Bi-miR-34a pull-down or down-regulated by miR-34a over-expression are enriched in growth factor signaling, cell cycle progression, and DNA repair pathways.
Network of canonical pathways (Wikipathways and KEGG) significantly enriched for genes identified by Bi-miR-34a pull-down (A) or down-regulated following miR-34a over-expression (B) in HCT116 and K562 cells. Each pathway is represented by a node in the network. The node size increases with the number of identified genes in the pathway and the node color represents the p-value based on the hypergeometric distribution (see key). Pathways that are not significantly enriched in an experiment are still shown, but are in gray. The number of genes shared between two pathways is represented by an edge whose thickness increases with the number of shared genes. The pull-down enriched pathways (A) suggest that miR-34a extensively targets growth factor, signal transduction and cell cycle control pathways. The integrated outcome of both direct and indirect effects of miR-34a on gene expression in (B) is suppression of expression of genes participating in downstream signaling, cell cycle and DNA repair pathways.
Figure 4
Figure 4. Interactome of genes in the enriched canonical pathways pulled down with Bi-miR-34a and/or down-regulated by miR-34a over-expression.
Interactome of products of genes identified by Bi-miR-34a pull-down (red) or down-regulated by miR-34a over-expression (blue) in significantly enriched pathways (Figure 3) in HCT116 cells. Edges represent physical interactions. A dense network of genes involved in growth factor signaling and downstream effects on cell cycle progression and DNA repair is implicated.
Figure 5
Figure 5. miR-34a expression suppresses cellular activation in response to serum growth factors.
(A,B) miR-34a over-expression reduces basal phosphorylation of AKT and ERK as measured by immunoblot 48 hr after transfection of HCT116 (A) and HeLa (B) cells with control (CTL) miRNA or miR-34a mimics. Abundance of total ERK and AKT doesn't change. (C,D) miR-34a over-expression reduces cell proliferation in the absence of serum and suppresses the proliferative response of HCT116 (C) and HeLa (D) cells 24 hr after adding serum. (E) Total numbers of miR-34a+/+ or miR-34a−/− MEFs after 24 hr of culture in 10% serum (10%) or 24 hr in 10% serum followed by 24 hr in 0.1% serum (0.1%). MEFs sufficient for miR-34a were more sensitive to serum starvation. (F) miR-34a+/+ MEFs were more prone to apoptosis than miR-34a−/− MEFs after 24 hr of culture in reduced serum. (G) Expression of miR-34 family members in miR-34+/+ (WT) and miR-34−/− MEFs assessed by qRT-PCR. miR-34a−/− MEFs expressed higher levels of miR-34b and miR-34c. In C,D,F, and G, data represent mean ± SD of 3 independent experiments. *, p<0.05, #, p<0.01, **, p<0.005, ##, p<0.001.
Figure 6
Figure 6. miR-34a directly inhibits growth factor signaling and signal transduction pathways by regulating novel genes.
(A) Five genes involved in growth factor signaling or signal transduction are pulled down by Bi-miR-34a. HCT116 cells were transfected with Bi-cel-miR67 (CTL miRNA, black) or Bi-miR-34a (white) mimics for 24 hr and mRNA capture was measured by qRT-PCR normalized to GAPDH. Five of 5 (ARAF, AXL, MEK1, MET and PIK3R2) genes identified by microarrays, but not housekeeping mRNAs SDHA and UBC, are significantly enriched in the Bi-miR-34a pull-down. (B) miR-34a decreases ARAF, AXL, MEK1, MET and PIK3R2 mRNAs, measured by qRT-PCR relative to GAPDH, in HCT116 cells transfected with CTL (black) or miR-34a (white) for 48 hr. UBC is a negative control gene. Relative mRNA levels were normalized to levels in CTL miRNA-transfected cells. (C) ARAF, AXL, MEK1, MET and PIK3R2 protein levels decline by immunoblot after miR-34a over-expression in HCT116 cells harvested 48 hrs after transfection with CTL miRNA or miR-34a mimic. Because of the low signal for AXL, an additional experiment probed for AXL with a longer exposure is shown at right. (D) miR-34a significantly regulates the 3′UTR of ARAF, AXL, MEK1 and MET in HeLa cells co-transfected with a dual luciferase reporter bearing the 3′UTR of each gene and CTL miRNA or miR-34a for 48 hr. Insertion of a sequence fully complementary to miR-34a into the Renilla luciferase 3′UTR (AS-34a) is the positive control. Luciferase activity was normalized to results obtained with the empty vector (V). (E) Luciferase reporters bearing PITA-predicted wild-type (WT) MREs from each target gene are significantly repressed in HeLa cells cotransfected with miR-34a. Point mutations (MT) that disrupt base pairing with miR-34a rescue reporter expression. MRE sequences are provided in Figure S4.
Figure 7
Figure 7. miR-34a pull-downs identify novel miR-34a targets involved in cell cycle progression.
(A) Bi-miR-34a pull-down captures transcripts of cell cycle genes. Enrichment of candidate mRNAs in HCT116 cells transfected with Bi-cel-miR-67 miRNA (CTL, black) or Bi-miR-34a (white) for 24 hr was assessed by qRT-PCR analysis normalized to GAPDH. SDHA and UBC mRNAs are housekeeping genes not enriched in the pull-downs. (B) miR-34a over-expression significantly suppresses mRNA levels of 5 of 7 cell cycle genes tested. mRNA expression was analyzed by qRT-PCR relative to GAPDH performed on total RNA harvested from HCT116 cells transfected with CTL miRNA (black) or miR-34a (white) mimics for 48 hr. Two candidate target genes (CCND3 and SMAD4) and the housekeeping genes SDHA and UBC are not significantly altered. (C) Protein levels of 6 of 6 cell cycle genes examined decrease with miR-34a over-expression. HCT116 cells were transfected with CTL miRNA or miR-34a mimics for 48 hr before immunoblot. β-Actin is a loading control. (D) miR-34a represses the 3′UTR of CCND3, MCM2, MCM5, PLK1 and SMAD4, but not MCM4 and MYT1. HeLa cells were cotransfected for 48 hr with CTL miRNA or miR-34a mimics and psiCHECK-2 empty vector (V) or psiCHECK-2 containing the 3′UTR of each gene in the Renilla luciferase 3′UTR. The positive control reporter contained a perfectly complementary sequence to miR-34a (AS-34a). Relative luciferase activity in miR-34a-transfected cells is normalized to CTL miRNA-transfected cells. (E) Dual luciferase reporter plasmids bearing wild-type (WT) MREs from CCND3, MCM2, MCM5 and SMAD4 are significantly repressed by co-transfection with miR-34a in HeLa cells. Mutation of the seed region of each MRE (MT) rescues reporter expression. MRE sequences are provided in Figure S5. *, p<0.05, #, p<0.01, **, p<0.005, ##, p<0.001.
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