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. 2006 Mar 20;34(5):1646-52.
doi: 10.1093/nar/gkl068. Print 2006.

Systematic identification of microRNA functions by combining target prediction and expression profiling

Xiaowei Wang  1 , Xiaohui Wang
Affiliations

Systematic identification of microRNA functions by combining target prediction and expression profiling

Xiaowei Wang et al. Nucleic Acids Res. .

Abstract

Target predictions and validations are major obstacles facing microRNA (miRNA) researchers. Animal miRNA target prediction is challenging because of limited miRNA sequence complementarity to the targets. In addition, only a small number of predicted targets have been experimentally validated and the miRNA mechanism is poorly understood. Here we present a novel algorithm for animal miRNA target prediction. The algorithm combines relevant parameters for miRNA target recognition and heuristically assigns different weights to these parameters according to their relative importance. A score calculation scheme is introduced to reflect the strength of each parameter. We also performed microarray time course experiments to identify downregulated genes due to miRNA overexpression. The computational target prediction is combined with the miRNA transfection experiment to systematically identify the gene targets of human miR-124. miR-124 overexpression led to a significant downregulation of many cell cycle related genes. This may be the result of direct suppression of a few cell growth inhibitors at the early stage of miRNA overexpression, and these targeted genes were continuously suppressed over a long period of time. Our high-throughput approach can be generalized to globally identify the targets and functions of other miRNAs.

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Figures

Figure 1
Figure 1
A simple flowchart for miRNA target prediction algorithm.
Figure 2
Figure 2
Real-time RT–PCR validation of the downregulated predicted miR-124 targets. The mRNA levels for downregulated targets at 4, 24 and 48 h were examined. The expression levels at 24 and 48 h are presented as percentages of that at 4 h. Among the validated targets, the microarray results indicated that genes 8801 and 8992 were first downregulated at 8 h; genes 8763, 3915, 6566, 9341, 10 449 and 10 634 were first downregulated at 16 h; genes 6836, 60 481 and 84 061 were first downregulated at 24 h; genes 27 230 and 55 225 were first downregulated at 32 h (downregulation is defined as at least 50% reduction of gene expression).
Figure 3
Figure 3
miR-124 overexpression time course analysis to identify predicted targets in total downregulated genes. (A) Counts of downregulated predicted targets at different time points after miR-124 transfections. (B) The percentages of predicted targets in total downregulated genes.
Figure 4
Figure 4
A boxplot to compare the prediction scores from all predicted miR-124 targets and from downregulated targets 24 h after transfection. The box in each panel represents the 50% range of each target list. The median scores were 33 and 54 for all human predicted targets and the downregulated targets, respectively.
Figure 5
Figure 5
Overlapping downregulated genes after 16 and 120 h of miR-124 overexpression. Thirteen genes were in both lists and seven of them were miR-124 targets. Overall, 76 predicted targets were represented on the arrays.
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