This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. MicroRNAs are small noncoding RNAs that regulate protein production of target mRNAs through recognition and binding of partially complementary sites located within the 3'untranslated region. The long-term goals of this research are: 1) identify miRNAs critical to inner ear development and homeostasis;2) establish the degree to which miRNAs dictate inner ear cell fate establishment and homeostasis;3) identify miRNA directed cellular pathways that intersect hearing disease etiology and implicate signaling pathways and/or novel avenues for potential therapeutic intervention. We developed preliminary data that show that the expression of a trio of miRNAs is significantly and specifically increased concomitant with mouse inner ear maturation. The hypothesis is that the upregulation of miRs 96, 182 and 183 is required for normal maturation and/or homeostasis through their contribution to downstream target gene regulation. Independent miRNA target gene prediction algorthims identify several potential target mRNAs known to be active participants in inner ear biology, including several transcription factors, signaling ligands and receptors.
Our aims are to: 1) establish the embryonic pattern of miR 96, 182, and 183 expression in the mouse ear;2) Determine if miRs 96, 182 and 183 inhibit expression of predicted target genes and affect the expression of hair cells marker genes using cell lines from early embryonic mouse inner ear and 3) begin to generate a novel transgenic mouse where endogenous levels of these miRNAs are missexpressed in either the entire ear or supporting cells to study the effects of miR 96,182 and 183 dysregulation in vivo. This pilot project focused on aim 3. The first experiment is to demonstrate that the miRNAs can be expressed in transgenic mice using the GFAP promotor, which is found in glial cells, including astrocytes, nonmyelinating Schwann cells and is expressed in maturing and differentiated supporting cells of the organ of Corti. Mouse embryos have been injected with a GFAP construct containing mouse pre-miR-183, -96 and -182, and birth of those embryos is anticipated March 6, 2007. If this is successful, the GFAP promoter will be used to target a conditional knockout of the miRNAs. Fulfillment of the stated objectives will provide novel reagents and a proof of principle that modulation of specific miRNA expression leads to direct effects on inner ear function, thus establishing a paradigm for novel miRNA mediated therapeutics. With the awarding of the F32 grant to fund this research, this one-year project will rotate off COBRE funding.
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