mRNAs through imperfect complementarities and reduce the level of target protein expression. Numerous studies have begun to reveal a critical and wide-spread role for microRNA in mammalian physiology and disease. The arguably most important yet challenging task in the field of microRNA research is experimental identification of specific targets of microRNA. It is relatively straightforward to identify microRNAs that are altered in a disease or experimental condition. But then investigators have to """"""""guess"""""""" from hundreds or more computationally predicted targets of each microRNA what the real targets might be. The very small number of verified microRNA-target pairs available and the inability to nominate likely microRNA-target pairs severely limits the entire field of microRNA research. The 3'-UTR reporter analysis provides the most specific evidence for a microRNA-target interaction. However, without high-probability candidates, performing the 3'-UTR reporter analysis for a large number of predicted interactions would be prohibitively costly with limited benefits. Our laboratory has developed a new approach to nominate high-probability, physiologically relevant microRNA-target pairs. It combines proteomic, microRNA expression, and bioinformatic analyses to identify microRNA-target pairs supported not only by computational prediction but also by reciprocal expression. The approach has been applied to identify 98 such pairs in kidney regions or a cell model of epithelial mesenchymal transition (EMT). The findings provide a unique basis that makes it practical to verify a large number of microRNA-target pairs using the 3'-UTR analysis. We propose to develop a high-throughput 3'- UTR assay to examine the 98 microRNA-target interactions as well as approximately 1,350 additional predicted interactions (Aim 1). Preliminary study has verified a microRNA-target interaction using the assay. We will perform a proof-of-concept study (Aim 2) to examine the functional significance of selected microRNA target pairs verified in Aim 1. Preliminary study has identified one such microRNA, the inhibition of which significantly attenuated transforming growth factor ?1-induced EMT. The proposed study will directly address a critical barrier in a rapidly emerging field and could have far-reaching implications. 1) The large number, possibly hundreds, of verified microRNA-target pairs will provide an extensive, unique, and much-needed resource for microRNA research in any subject area, particularly in renal physiology and disease. 2) The study will allow us to evaluate and possibly improve various approaches for nominating microRNA-target pairs, providing methodological advances critical to the field of microRNA research. 3) The study may reveal novel mechanistic insights into the role of specific microRNAs in EMT and renal fibrosis that may be further investigated in future studies.
The result of the study in this R21 proposal may greatly facilitate future studies of microRNA in human health and disease and provide novel insights into the mechanisms underlying the development of chronic renal injury.
