Differential inhibition of target gene expression by human microRNAs

Abstract

microRNA (miRNA) target genes are commonly computationally predicted, yet how dependable predictions are remains unclear, as most predicted target genes have not been rigorously tested experimentally. In addition, the degree to which a miRNA differentially inhibits the expression of its targets is underappreciated. We selected human miR‐1, miR‐122, and miR‐124 as representatives to investigate the reliability of miRNA target predictions and to examine how miRNAs suppress their target genes. We first constructed target gene reporter libraries for the miRNAs based on three prediction programs: TargetScan, miRanda, and PicTar, and used reporter assays in cell cultures to directly evaluate whether as well as how strongly a predicted target gene is repressed by its cognate miRNA. We found that all three programs have approximately 72–85% success rates in predicting genuine miRNA targets, and that miRNAs inhibit different targets to various degrees. We then performed statistical and correlation analyses to examine how well parameters offered by the online programs and features in target mRNAs might predict the degrees of repression by miRNAs. To show that endogenous miRNAs act through the same mechanism, we correlated our reporter assay results with publically available mRNA expression data in vivo. Overexpression of miR‐1, miR‐122, or miR‐124 further led to differential reduction of target mRNA levels in cell cultures. Our studies systematically investigated hundreds of miRNA target genes, shed light onto the performance of miRNA target gene prediction programs, and suggested a new mechanism by which differential target repression by miRNAs regulates gene expression in vivo.Support or Funding InformationThis work was supported in part by the National Natural Science Foundation of China [grant number 31570843].