An IGF/miR483-3p Regulatory Loop Influences Human Placental Development

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 88-108-GHRH, GH & IGF Biology & Signaling
Basic/Translational
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-101
Farkhondeh Farrokhnia, Richard Unwin, Adam Stevens and Karen Forbes*
University of Manchester, Manchester, United Kingdom
Fetal growth complications such as fetal growth restriction (FGR) and fetal overgrowth (macrosomia) are associated with altered placental growth and function. Insulin-like growth factors (IGFs) are required for optimal placental cell (cytotrophoblast; CT) proliferation, thus they are important mediators of both placental and fetal growth. We have recently demonstrated that microRNAs (miRs) are also important regulators of CT proliferation. In this study, we explored the possibility that IGFs and miRs interact to regulate placental growth.  

First trimester placental explants were cultured in serum-free conditions for 24h and then exposed to IGF-I (10nM) for a further 24h (n=6). miR expression was profiled using arrays and revealed that IGF-I significantly reduced expression of two miRs (P<0.001). In-situ hybridisation revealed that one of these; miR-483-3p, was expressed within CT in first trimester placenta suggesting that it may influence events in these cells.  To determine the role of miR-483-3p in CT, negative control or miR-483-3p specific inhibitors (30-100nM) were introduced into placental explants by nucleofection and following confirmation of miR inhibition (QPCR), levels of proliferation (Ki67 and BrdU) were assessed by immunohistochemistry.  Analysis of CT proliferation revealed that inhibition of miR-483-3p expression significantly enhanced levels of CT proliferation (P< 0.01; n=6). To determine the gene targets of miR-483-3p, an iTRAQ based proteomics strategy was performed and revealed that several proteins were altered in trimester placental explants with low miR-483-3p expression (miR-483-3p inhibitor) compared to control tissue (negative control inhibitor). Network analysis was used to identify potential biological pathways and revealed enrichment for growth and development related proteins.  Furthermore, IGF-I and ELAVL1/HuR were identified as key regulatory proteins that are influenced by miR-483-3p.  Western blotting and QPCR confirmed that miR-483-3p negatively regulates IGF-I and ELAVL1/HuR expression, and 3’UTR luciferase reporter and anti-Agonaute RIP assays demonstrated that the interactions are direct. 

Here we demonstrate that maternal IGF-I acts via miR-483-3p to enhance placental IGF-I and ELAVL1 expression. This positive regulatory loop appears to influence CT proliferation and placental growth.  Although the role of ELAVL1/HuR in the human placenta remains to be established, in mouse placenta it regulates the cleavage of the growth regulatory miR, miR-675 from the H19 locus of the igf2/H19 gene.  Interestingly miR-483-3p is located the igf2 locus of the igf2/H19 gene. A balance between igf2 and H19 expression is crucial for proper embryogenesis and placental growth thus ongoing work is examining whether placental growth is controlled by an IGF-I --> igf2/miR483-3p -->IGF-I/ELAVL1/HuR --> H19/miR-675 regulatory pathway.

Nothing to Disclose: FF, RU, AS, KF

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: KF is supported by a University of Manchester Stepping Stone Fellowship