SUN-475 Analysis of Palmitate- and Bisphenol A-Mediated Changes in MicroRNAs Targeting the Novel Reproductive Peptide Phoenixin and Its Receptor Gpr173 in Hypothalamic Cell Lines

Abstract

Phoenixin (PNX) is a recently identified peptide that forms part of the hypothalamic-pituitary-gonadal axis. It is a highly conserved, 14 or 20 amino acid peptide expressed at high levels in the hypothalamus. Within hypothalamic neurons, PNX increases gene expression of gonadotropin-releasing hormone (GnRH) and kisspeptin, two critical reproductive peptides. In relation to reproductive function, knockdown of either PNX or its receptor, GPR173, extends the estrous cycle. As PNX and GPR173 are relatively understudied, delineating how this peptide and its putative receptor are regulated at the gene level is essential for further definition of functional significance. We have previously reported that palmitate, a saturated fatty acid, increases Pnx and reduces Gpr173 mRNA levels over 24 hours in immortalized hypothalamic clonal cell lines. We have also found that bisphenol A (BPA), an endocrine disrupting chemical widely thought to inhibit reproductive function, reduces Pnx and Gpr173 gene expression. The mechanism by which these compounds alter gene expression is the subject of current investigation. One layer of gene regulation is microRNAs (miRNAs), short segments of RNA that decrease mRNA expression levels. Palmitate and BPA are known to alter microRNA (miRNA) levels in tissues outside of the hypothalamus; therefore, we hypothesized that levels of miRNAs that target Pnx and Gpr173 in the hypothalamus may be contributing to the observed palmitate- and BPA-mediated changes in mRNA levels. Using the predictive online resources, miRwalk 3.0, TargetScan and miRanda, we have identified 112 miRNAs with putative binding sites in the Pnx 3’UTR, as well as 300 miRNAs that can bind the Gpr173 3’UTR. To identify miRNAs affected by palmitate or BPA exposure, the mHypoE-46 and the mHypoA-59 cell lines were treated with 50 μM palmitate or 100 μM BPA, respectively, for 4 and 24 hours. The miRNA profiles after these treatments are being assessed on the Affymetrix GeneChip miRNA 4.0 array, which includes over 30,000 probe sets for mature miRNAs. Whether the miRNAs mediate the alterations in Pnx and Gpr173 gene expression will be assessed through the use of antagomirs and mimics. Knowledge of miRNAs controlling Pnx and Gpr173 expression contributes to our understanding of how nutrients and environmental chemicals affect the hypothalamus, potentially representing new targets for therapeutics.