MicroRNAs (miRNAs) are important players of post-transcriptional gene regulation. Individual miRNAs can target multiple mRNAs and a single mRNA can be targeted by many miRNAs. We hypothesized that miRNAs select and regulate their targets based on their own expression levels, those of their target mRNAs and triggered feedback loops. We studied the effects of varying concentrations of let-7a-7f and the miR-17-92 cluster plasmids on the reporter genes carrying either DICE R- or cMYC -3'UTR in Huh-7 cells. We showed that let-7 significantly downregulated expression of DICE R 3'UTR reporter at lower concentrations, but selectively downregulated expression of a cMYC 3'UTR reporter at higher dose. This miRNA dose-dependent target selection was also confirmed in other target genes, including CC ND1, CDKN1 and E2F1. After overexpressing let-7a-7f or the miR-17-92 clusters at wide-ranging doses, the target genes displayed a nonlinear correlation to the transfected miRNA. Further, by comparing the expression levels of let-7a and miR-17-5p, along with their selected target genes in 3 different cell lines, we found that the knockdown dose of each miRNA was directly related to their baseline expression level, that of the target gene and feedback loops. These findings were supported by gene modulation studies using endogenous levels of miR-29, -1 and -206 and a luciferase reporter system in multiple cell lines. Finally, we determined that the miR-17-92 cluster affected cell viability in a dose-dependent manner. In conclusion, we have shown that miRNAs potentially select their targets in a dose-dependent and nonlinear fashion that affects biological function; and this represents a novel mechanism by which miRNAs orchestrate the finely tuned balance of cell function.
Bibliographical noteFunding Information:
We thank members of both the C.J.S. and S.S. laboratories for their technical assistance and comments on the manuscript. This work was supported by National Institutes of Health ARRA Grant R01 DK081865-01 (to C.J.S.); Department of Defense Grant W81XWH-10-1-0556; and University of Minnesota Academic Health Center and Minnesota Medical Foundation (to S.S.).
- MiRNA target selection
- Tumor suppressor gene