Heterogeneous nuclear ribonucleoprotein C1 may control miR-30d levels in endometrial exosomes affecting early embryo implantation.

Is there a specific mechanism to load the microRNA (miRNA), hsa-miR-30d, into exosomes to facilitate maternal communication with preimplantation embryos? The heterogeneous nuclear ribonucleoprotein C1 (hnRNPC1) is involved in the internalization of endometrial miR-30d into exosomes to prepare for its subsequent incorporation into trophectoderm cells. Our group previously described a novel cell-to-cell communication mechanism involving the delivery of endometrial miRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. Specifically, human endometrial miR-30d is taken up by murine blastocysts causing the overexpression of certain genes involved in embryonic adhesion (Itb3, Itga7 and Cdh5) increasing embryo adhesion rates. Transfer of maternal miR-30d to preimplantation embryos was confirmed by co-culture of wild-type (WT) and miR-30d knockout (KO) murine embryos with primary cultures of human endometrial epithelial cells (hEECs) in which mir-30d was labeled with specific Molecular Beacon (MB) or SmartFlare probes. Potential molecules responsible for the miR-30d loading into exosomes were purified by pull-down analysis with a biotinylated form of miR-30d on protein lysates from human endometrial exosomes, identified using mass spectrometry and assessed by flow cytometry, western blotting and co-localization studies. The role of hnRNPC1 in the miR-30d loading and transportation was interrogated by quantification of this miRNA in exosomes isolated from endometrial cells in which hnRNPC1 was transiently silenced using small interference RNA. Finally, the transfer of miR-30d to WT and KO embryos was assessed upon co-culture with sihnRNPC1 transfected cells. Murine embryos from miR-30d WT and KO mice, (strain MirC26tm1Mtm/Mmjax), were obtained by oviduct flushing of superovulated females. Endometrial Exosomes were purified by ultracentrifugation of supernatants from primary cultures of hEECs or Ishikawa cells. MB and Smartflare miR-30d probes were detected by confocal and/or transmission electron microscopy (TEM). hEECs and exosomes derived from them were subjected to pull-down with a biotinylated form of miR-30d. Captured proteins were identified by mass spectrometry (MS/MS). Western blotting was performed to detect hnRNPC1 and CYR61 in whole lysates, subcellular fractions and secreted vesicles from hEECs. Co-localization studies of the selected proteins with the exosomal marker CD63 were performed. FACS analysis was carried out to determine the presence of hnRNPC1 inside exosomes. Silencing of hnRNPC1 was conducted in the Ishikawa Cell Line with the Smart Pool Accell HNRNPC siRNA at a final concentration of 50 nM. RT-qPCRs were done to determine the messenger levels of miR-30d in cells and exosomes. Co-cultures of WT and KO embryos were established with Ishikawa cells double-transfected with sihnRPNC1 and MB probes. MS/MS analysis allowed us to identify hnRNPC1 as a possible protein to influence miR-30d loading into exosomes. Co-localization studies of hnRNPC1 with CD63 and FACS analyses suggested the presence of hnRNPC1 inside exosomes. Silencing of hnRNPC1 in Ishikawa cells resulted in a sharp decrease of the levels of miR-30d in both epithelial-like cells (P = 0.0001) and exosomes (P = 0.0152), suggesting its potential role in miR-30d biogenesis and transfer. Co-culture assays of miR-30d KO embryos with sihnRNPC1 hEECs revealed a decrease in embryo-miR-30d acquisition during the adhesion and invasion stages. In turn, transient silencing of hnRNPC1 results in a significant decrease of blastocyst adhesion compared to mock transfection conditions using Block-it, in both WT [Mean ± SD; 67 ± 10.0% vs. 38 ± 8.5%(P = 0.0006)] and miR-30d KO embryos [Mean ± SD; 50 ± 11.5% vs. 26 ± 8.8% (P = 0.0029) (n = 2); 14 embryos transferred per condition tested]. MS/MS data are available via ProteomeXchange with identifier PXD008773. The Ishikawa Cell Line was used as a model of hEECs in silencing experiments due to the low survival rates of primary hEECs after transfection. The data show that hnRNPC1 may be involved in the internalization of miR-30d inside exosomes. The decreased rates of embryo adhesion in endometrial epithelial-like cells transiently silenced with sihnRNPC1evidence that hnRNPC1 could be an important player in the maternal-embryo communication established in the early stages of implantation. This work was supported by the Miguel Servet Program Type I of Instituto de Salud Carlos III [CP13/00038]; FIS project [PI14/00545] to F.V.; the 'Atracció de Talent' Program from VLC-CAMPUS [UV-INV-PREDOC14-178329 to NB]; a Torres-Quevedo grant (PTQ-13-06133) by the Spanish Ministry of Economy and Competitiveness to IM and MINECO/FEDER Grant [SAF2015-67154-R] to C.S. The authors declare there is no conflict of interest.