Identification and Characterization of Avian Pleiotrophin as Estrogen-Stimulated Gene in the Chicken Oviduct and Its Differential Expression Patterns in Cancerous Ovaries.

Abstract

Pleiotrophin (PTN), also known as heparin-binding growth factor 8 (HBGF-8), is a developmentally-regulated growth factor expressed in normal tissues and in carcinomas. Although PTN is involved in regulation of cellular development and differentiation and the etiology of carcinogenesis, little is known about its expression and regulation in either the oviduct or normal and cancerous ovaries of chickens. This novel study compared chicken and mammalian PTN genes with respect to structure, phylogenetic evolution, tissue specific expression of PTN mRNA and protein, and regulation of expression by estrogen in the chicken oviduct. We performed RT-PCR, quantitative RT-PCR, in situ hybridization and immunohistochemical analyses to detect PTN mRNA expression and protein localization in organs of adult hens, young chicks treated with diethylstilbestrol (DES) and laying hens with ovarian carcinoma. Chicken PTN is highly conserved with respect to mammalian PTN genes (91-92.6%) and its mRNA expression was most abundant in brain, heart and oviduct. This study focused on the avian PTN gene in the female chicken reproductive organs. Treatment of young chicks with diethylstilbestrol induced PTN mRNA and protein only in luminal and glandular epithelia of the oviduct. Further, PTN gene expression is influenced by several micro RNAs, specifically miR-499 and miR-1709 through its 3'-UTR, which suggests that post-transcriptional regulation influences PTN expression in chickens. We also compared CpG methylation status of the PTN gene in normal and cancerous ovaries from laying hens to discover high expression of the PTN gene in glandular epithelia of ovarian carcinoma in laying hens with 30- and 40% of -1311 and -1339 CpG sites being demethylated in ovarian cancer cells, but not in normal ovarian epithelial cells. In conclusion, chicken PTN is a novel estrogen-stimulated gene expressed abundantly in oviductal epithelia implicating roles in oviduct development and egg formation. PTN is also a biomarker for epithelial ovarian carcinoma that could be used for diagnosis and monitoring effects of cancer therapies. Research supported by the World Class University (WCU) program (R31-10056) and by Basic Science Research Program (2010-0013078) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology.