Two-tiered approach identifies a network of cancer and liver disease-related genes regulated by miR-122 - PubMed (original) (raw)

. 2011 May 20;286(20):18066-78.

doi: 10.1074/jbc.M110.196451. Epub 2011 Mar 14.

Patrick J Collins, Uthra Suresh, Mingzhu Lu, Cristina M Ramírez, Carlos Fernández-Hernando, Yufei Huang, Raquel de Sousa Abreu, Shu-Yun Le, Bruce A Shapiro, Angela M Liu, John M Luk, Shelley Force Aldred, Nathan D Trinklein, Edward M Marcotte, Luiz O F Penalva

Affiliations

Daniel R Boutz et al. J Biol Chem. 2011.

Abstract

MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.

© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Luciferase-based screening to identify miR-122 targets. SwitchGear luciferase reporter constructs containing 3′UTRs of 139 genes predicted to be miR-122 target sites (blue ♦) were assayed for repression in response to miR-122 treatment. Negative controls (red ■) consisted of 11 3′UTRs from human genes lacking predicted miR-122 target sites and five scrambled sequence controls. An additional 14 genes (yellow ▴) identified as putative targets in the proteomic analysis were also assayed. Constructs exhibiting greater than 1.5-fold repression (log2 − ratio < −0.58) and a p value <0.05 (log10(p value) < −1.3) (miR-122-treated versus control) were considered significantly down-regulated (bottom-left quadrant).

FIGURE 2.

FIGURE 2.

Proteomic analysis identifies down-regulated proteins with strong enrichment for miR-122 target site prediction. Predicted target sites were identified within 3′UTRs of proteins down-regulated in our proteomic analysis using various target prediction algorithms as follows: seed site complementarity of 7-mer-A1 and 7-mer-m8 motifs, 8-mer, and at either 7- or 8-mer (7–8-mer) seeds, as well as the TargetScan 5.1 with strict conservation of target sites (TS-Con). Results were plotted as the occurrence of a predicted target site versus the ranking of the proteins based on increasing down-regulation (A), and level of enrichment as the ratio of the frequency of predicted targets within each subset versus the total experimental dataset (B).

FIGURE 3.

FIGURE 3.

Summary of the combined down-regulated proteomics and luciferase datasets. A combined 260 genes were identified as significantly down-regulated in at least one of two experimental approaches. Significance was defined as p value < 0.05 and fold-change >1.5 for luciferase experiments and Z-score >1.65 and fold-change >1.3 for proteomics experiments. Direct refers to significantly down-regulated genes containing a 7-mer or greater miR-122 seed complementary site; Indirect refers to significantly down-regulated genes lacking such a site.

FIGURE 4.

FIGURE 4.

Identified targets show miR-122-induced repression of protein levels in Huh-7 cells. A, Western blots of five proteins identified as targets in our screen, from control mock-transfected (CM) and miR-122-transfected Huh-7 cells. B, relative protein abundance levels plotted from Western data show significant down-regulation of all five target proteins tested. VIM, vimentin.

FIGURE 5.

FIGURE 5.

Mutagenesis of miRNA seed recognition sequence disrupts miR-122 repression. Six different genes identified as targets of miR-122 were selected for a mutagenesis study to partially validate our analysis and determine the importance of the seed sequence in miR-122 function. Two to three nucleotides were altered in the 3′UTR region of each gene matching the seed sequence and analyzed in luciferase assays. * designates a p value < 0.005; ** designates a p value <0.005. Mut, mutant; ALDOA, aldolase A.

FIGURE 6.

FIGURE 6.

Consensus binding motifs calculated for targets identified by luciferase and proteomics analyses. The probability of involvement of a nucleotide in base pairing is displayed as the height of the corresponding letter for the miR-122 sequence (A) and predicted mRNA target site (B). Binding motifs were determined for the proteomics datasets. Binding motifs for the luciferase dataset show a very similar trend (not shown).

FIGURE 7.

FIGURE 7.

Gene ontology analysis of miR-122 identified targets. Pathway Studio 6 (Ariadne Genomics) was used to determine enrichment of cellular processes in the combined luciferase/proteomics down-regulated direct target set (dark gray bars). Total human transcriptome was used as background (light gray bars). * designates a p value <0.05; ** designates a p value <0.005.

FIGURE 8.

FIGURE 8.

miR-122 and liver-related functions and diseases. The association between identified miR-122 targets and liver-related processes and diseases was investigated with Pathway Studio 6. Green indicates the genes identified in the proteomics analysis containing at least one miR-122 seed sequence; red indicates the genes identified by the luciferase analysis; blue indicates the genes identified by both methods. Liver function and disease associations are displayed in purple. ALDOA, aldolase A; Vim, vimentin; CS, citrate synthase; PXN, paxillin.

FIGURE 9.

FIGURE 9.

Associations among miR-122-identified targets. Pathway Studio 6 was used to establish connections among genes that responded to miR-122. Genes identified in the proteomics analysis as direct miR-122 targets containing predicted target sites are represented in green, and indirect targets lacking such sites are brown; pink indicates targets identified by the luciferase analysis; and blue indicates high confidence targets identified by both methods. PXN, paxillin.

References

    1. Lee R. C., Feinbaum R. L., Ambros V. (1993) Cell 75, 843–854 - PubMed
    1. Griffiths-Jones S., Saini H. K., van Dongen S., Enright A. J. (2008) Nucleic Acids Res. 36, D154–D158 - PMC - PubMed
    1. Bartel D. P. (2004) Cell 116, 281–297 - PubMed
    1. Filipowicz W., Bhattacharyya S. N., Sonenberg N. (2008) Nat. Rev. Genet. 9, 102–114 - PubMed
    1. Kiriakidou M., Nelson P. T., Kouranov A., Fitziev P., Bouyioukos C., Mourelatos Z., Hatzigeorgiou A. (2004) Genes Dev. 18, 1165–1178 - PMC - PubMed

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