title: Dose-dependent differential mRNA target selection and regulation by let-7a-7f and miR-17-92 cluster microRNAs.
abstract: 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 DICER- or cMYC -3'UTR in Huh-7 cells. We showed that let-7 significantly downregulated expression of DICER 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 CCND1, 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.
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA.
authors.forename: Jingmin
authors.initials: J
authors.lastname: Shu
authors.forename: Zhilian
authors.initials: Z
authors.lastname: Xia
authors.forename: Lihua
authors.initials: L
authors.lastname: Li
authors.forename: Ellen T
authors.initials: ET
authors.lastname: Liang
authors.forename: Nicholas
authors.initials: N
authors.lastname: Slipek
authors.forename: Dawen
authors.initials: D
authors.lastname: Shen
authors.forename: Jasmine
authors.initials: J
authors.lastname: Foo
authors.forename: Subbaya
authors.initials: S
authors.lastname: Subramanian
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Steer
mesh_terms: D020413:3' Untranslated Regions; D045744:Cell Line, Tumor; D002470:Cell Survival; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D062085:RNA, Long Noncoding; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; C530104:CCND1 protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C568997:MIR17HG, human; C489427:MYC protein, human; D035683:MicroRNAs; D016271:Proto-Oncogene Proteins c-myc; D062085:RNA, Long Noncoding; C487931:mirnlet7 microRNA, human; D019938:Cyclin D1; D008156:Luciferases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases