Abstract
MicroRNAs (miRs) target and inhibit complementary messenger RNAs (mRNAs). The miR-183 family (miR-183, -182. and -96) is already known to aid in the development and upkeep of hair cells in the inner ear. Hair cells convert mechanosensory sound waves to nerve impulses. They develop in conjunction with supporting cells, where the precise regulation of gene expression reinforces differentiation as either a hair cell or supporting cell. miRs inhibit gene expression through interaction of their 7 base pair "seed sequence" with the 3' untranslated regions (3' UTRs) of complimentary mRNAs. Because the sequences of the miRs are known, the interactions of miRs with target genes are easily predicted. As such, there are databases of proposed interactions that require experimental validation. The 3' UTRs of predicted miR-183 target genes Sox2, Notch 1, Hesl, and Jagl were inserted into a luciferase reporter vector and transfected into cultured HEK293 cells with control RNA or miR-183 family members. After 24 hours, cell lysates were collected and analyzed to quantify luciferase activity. The reporter vector contains the 3' UTR downstream of a Photinous luciferase (Pluc) gene. The vector also contains the gene for Renilla luciferase (Rluc). miR binding to the 3'UTR is expected to decrease Pluc expression relative to RJuc expression in a dual luciferase assay. Analysis of the data showed downregulation of the Sox2 reporter gene by miR-182 but not miR-183 or -96, consistent with a predicted miR-182 binding site. Similarly. Hesl reporter gene was downregulated with miR-182 and -96 but not miR-183, consistent with a predicted miR-182/96 binding site. The Notchl reporter gene showed downregulation with each individual miR-183 family member and greater downregulation with all three miRs combined, consistent with each miR having a predicted binding site. Jagl reporter gene showed no downregulation with any miR-183 family member despite having a weak predicted miR-183 binding site. These results support the conclusion that miR-183 family members downregulate genes that are associated with supporting cell development, thus reinforcing hair cell fate.