The Mir-200 Family and Their Targets Are Hormonally Regulated in Endometrial Stromal Cells and Likely Serve a Key Role in Blastocyst Implantation

Presentation Number: SUN-0043
Date of Presentation: June 22nd, 2014

Patricia Tereese Jimenez*1, Sarah White1 and Carole R Mendelson2
1University of Texas Southwestern Medical Center, Dallas, TX, 2UT Southwestern Med Ctr, Dallas, TX

Abstract

Endometrial stromal cells are hormonally responsive and must undergo molecular, structural and functional changes for the uterus to become receptive for blastocyst implantation and to support embryo development. microRNAs (miRNA/miR) and their targets have been proposed to serve regulatory roles in implantation. To define miRNAs that are differentially regulated in endometrial stroma during implantation, we conducted miRNA microarray analysis of 1281 mature miRNAs in stromal cells of mouse uterus at 0.5 days post-coitum (dpc, prior to implantation) and from implantation sites at 4.5 dpc (during implantation). Intriguingly, we observed that all members of the evolutionarily conserved miR-200 family were downregulated more than two-fold at 4.5 vs. 0.5 dpc. TGFβ and transcription factors ZEB1 and ZEB2, confirmed targets of miR-200s, are known positive regulators of epithelial-to-mesenchymal transition (EMT) and stimulate metastasis and invasion of cancer cells. miR-200s and ZEBs exist in a double-negative feedback loop, whereas, ZEBs and TGF-β positively regulate one another. To confirm and extend results of the microarray, RT-qPCR of RNA from endometrial stromal cells isolated from a separate cohort of mice at 0.5, 2.5, 4.5 and 8.5 dpc revealed that between 0.5 and 2.5 dpc, miR-200a, -200b and -200c were significantly downregulated, while ZEB1/2 and TGF-β, were significantly, but transiently, upregulated. N-cadherin, a known mesenchymal marker, was expressed in a similar pattern as ZEB1/2 and TGF-β, while the epithelial marker, E-cadherin, was increased between 4.5 dpc and 8.5 dpc, in association with decidualization. These findings suggest that EMT and the reverse process, MET, may serve important roles in implantation, invasion and decidualization. To analyze hormonal regulation of the miR-200 family and targets, ovariectomized mice were subcutaneously injected with vehicle, 1 mg 17β estradiol (E2) or 1 mg progesterone (P4) for 1, 2 or 3 days. Endometrial stromal cells were isolated from whole uteri and analyzed by RT-qPCR. Notably, miR-200 family members were markedly upregulated by E2 at each time point. On the other hand, ZEB1 and ZEB2 were downregulated by E2 and upregulated by P4 following 2 days of treatment. Our collective findings suggest that miR-200s and their targets serve as hormonally-modulated mediators of the coordinated signaling pathways during embryo implantation. Furthermore, miR-200-ZEB-TGF-β regulation of EMT/MET within endometrial stroma may be critical to successful implantation and decidualization.

 

Nothing to Disclose: PTJ, SW, CRM