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. 2009 Mar;19(3):481-90.
doi: 10.1101/gr.084129.108. Epub 2008 Dec 16.

MicroRNA Target Prediction by Expression Analysis of Host Genes

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Free PMC article

MicroRNA Target Prediction by Expression Analysis of Host Genes

Vincenzo Alessandro Gennarino et al. Genome Res. .
Free PMC article

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression by inducing RNA cleavage or translational inhibition. Most human miRNAs are intragenic and are transcribed as part of their hosting transcription units. We hypothesized that the expression profiles of miRNA host genes and of their targets are inversely correlated and devised a novel procedure, HOCTAR (host gene oppositely correlated targets), which ranks predicted miRNA target genes based on their anti-correlated expression behavior relative to their respective miRNA host genes. HOCTAR is the first tool for systematic miRNA target prediction that utilizes the same set of microarray experiments to monitor the expression of both miRNAs (through their host genes) and candidate targets. We applied the procedure to 178 human intragenic miRNAs and found that it performs better than currently available prediction softwares in pinpointing previously validated miRNA targets. The high-scoring HOCTAR predicted targets were enriched in Gene Ontology categories, which were consistent with previously published data, as in the case of miR-106b and miR-93. By means of overexpression and loss-of-function assays, we also demonstrated that HOCTAR is efficient in predicting novel miRNA targets and we identified, by microarray and qRT-PCR procedures, 34 and 28 novel targets for miR-26b and miR-98, respectively. Overall, we believe that the use of HOCTAR significantly reduces the number of candidate miRNA targets to be tested compared to the procedures based solely on target sequence recognition. Finally, our data further confirm that miRNAs have a significant impact on the mRNA levels of most of their targets.

Figures

Figure 1.
Figure 1.
Flowchart of the HOCTAR procedure (see text for further details).
Figure 2.
Figure 2.
HOCTAR performance in recognizing previously validated miRNA targets. Comparison of HOCTAR with three sequence-based miRNA target prediction softwares (miRanda, TargetScan, and PicTar) in predicting 56 validated targets of 20 different miRNAs (see text for further details). (A) The panels display, for each of the algorithms analyzed, the ranking position, in a percentile format, of the validated targets selected for the analysis (orange horizontal lines). The distribution of the predictions by HOCTAR tends to be shifted toward the top 50th percentile in comparison to the other three softwares. (B) Summary of the number of validated targets predicted by each of the algorithms.
Figure 3.
Figure 3.
Experimental validation of a subset of HOCTAR predictions by overexpression of mimic-microRNAs in HeLa cells, as assessed by qRT-PCR. Histograms showing differences in the expression levels, between miRNA-overexpressed HeLa cells (black bars) and control (cel-miR-67 transfected) HeLa cells (white bars), of a subset of HOCTAR predicted target genes for miR-26b (A) and miR-98 (B) and of a subset of control genes by qRT-PCR assays. Y-axis: fold change repression (expressed as 2−ΔΔCt values). X-axis: predicted target genes tested (their symbols are indicated in the light blue boxes). The target genes tested were distributed along the entire HOCTAR ranked prediction lists for miR-26b (A) and miR-98 (B), and the red lines below the diagrams indicate the ranking of each target genes tested within the HOCTAR prediction ranked list for miR-26b (A) and miR-98 (B). The control genes shown on the right part of the two diagrams are not predicted to represent targets of the two analyzed miRNAs. The housekeeping (HK) genes used to normalize the expression of genes are HPRT1 and GAPDH. The analysis of each gene tested was performed in triplicate.
Figure 4.
Figure 4.
Genes down-regulated after miR-26b and miR-98 overexpression are overrepresented in high-scoring HOCTAR predictions, as determined by microarray analysis. Enrichment plots generated by GSEA analysis of the HOCTAR predictions list for miR-26b and miR-98 are represented in A and B. The enrichment score is shown as a green line in each plot, and the vertical black bars below the plots indicate the position of probes belonging to the HOCTAR prediction lists for miR-26b within the rank ordering of the 22,277 probes present on the human U133A microarray from the probe most down-regulated (position 1 on the left) to the most up-regulated in HeLa cells after miR-26b (A) and miR-98 (B) transfection. (C,D) Histograms showing the number of probes with significant expression changes in HeLa cells after miR-26b (C) and miR-98 (D) overexpression (size, Y-axis). Each bar represents one of the 10 bins (probe sets, X-axis) in which the entire HOCTAR lists for miR-26b and miR-98 were subdivided (see the text for further details).
Figure 5.
Figure 5.
Experimental validation of the HOCTAR procedure by down-regulation of miR-26b and miR-98 in HeLa cells. Histogram showing differences in the expression levels between HeLa cells transfected with a miRNA-inhibitor (black bars) and control (cel-miR-67 transfected) HeLa cells (white bars), of a subset of HOCTAR predicted target genes for miR-26b (A) and miR-98 (B) and of a subset of control genes by qRT-PCR assays. Y-axis: fold change activation (expressed as 2−ΔΔCt values). X-axis: predicted target genes tested (their symbols are indicated in the light blue boxes). The target genes tested were distributed along the entire HOCTAR ranked prediction lists for miR-26b (A) and miR-98 (B) and the red lines below the diagrams indicate the ranking of each target genes tested within the HOCTAR prediction ranked list for miR-26b (A) and miR-98 (B). The control genes shown on the right part of the two diagrams are not predicted to represent targets of the two analyzed miRNAs. The housekeeping (HK) genes used to normalize the expression of genes are HPRT1 and GAPDH. The analysis of each gene tested was performed in triplicate.

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