Investigating the role of mir-190, mir-200, and mir-8 inhibition of the GCS protein in HeLa cells and Drosophila melanogaster

Glucosylceramide synthase (GCS or GlcT-1 in Drosophila and UGCG in humans) is a key enzyme in sphingolipid metabolism and obesity. Pharmacological inhibition of GCS in obese mice improves their insulin responsiveness, adipocyte functions, and also reduces inflammation and liver fat content. MicroRNAs are non-coding RNAs that specifically inhibit genes by binding to target sites in the gene's mRNA. Based on preliminary evidence that GCS is regulated by microRNAs in vitro and that it contains well conserved binding sites for the microRNAs mir-8, mir-190, and mir-200, we propose to investigate the role of these microRNAs in GCS regulation and their ability to inhibit this protein. We propose to investigate the role of these microRNAs both in human HeLa cells and in vivo in Drosophila melanogaster. In order to do this, we began by transfecting human HeLa cells with mir-190 and mir-200 as well as over-expressing mir-8 and mir-190 in Drosophila melanogaster using the UAS-Gal4 system. We also included negative microRNAs (untranfected cells and normal flies) as controls. After transfection and over-expression, the RNA was isolated and reverse transcribed into cDNA. This cDNA was then amplified and the effects of the microRNAs on the GCS protein were measured using qPCR.