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

STUDY QUESTION: Is there a specific mechanism to load the microRNA (miRNA), hsa-miR-30d, into exosomes to facilitate maternal communication with pre-implantation embryos? A SUMMARY ANSWER: The heterogeneous nuclear ribonucleoprotein CI (hnRNPC I ) is involved in the internalization of endometrial miR-30d into exosomes to prepare for its subsequent incorporation into trophectoderm cells. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: Transfer of maternal miR-30d to preimplantation embryos was confirmed by co-culture of wildtype (WT) and miR-30d knockout (KO) murine embryos with primary cultures of human endometrial epithelial cells (hEECs) in which mir30d 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 hnRNPCI in the miR-30d loading and transportation was interrogated by quantification of this miRNA in exosomes isolated from endometrial cells in which hnRNPCI was transiently silenced using small interference RNA. Finally, the transfer of miR-30d to WT and KO embryos was assessed upon co-culture with sihnRNPCI transfected cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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 hnRNPCI and CYR6 I 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 hnRNPCI inside exosomes. Silencing of hnRNPCI 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 sihnRPNCI and MB probes. MAIN RESULTS AND THE ROLE OF CHANCE: MS/MS analysis allowed us to identify hnRNPCI as a possible protein to influence miR-30d loading into exosomes. Co-localization studies of hnRNPCI with CD63 and FACS analyses suggested the presence of hnRNPC I inside exosomes. Silencing of hnRNPCI 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 sihnRN PC I hEECs revealed a decrease in embryo-miR-30d acquisition during the adhesion and invasion stages. In turn, transient silencing of hnRN PC I 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]. LARGE-SCALE DATA: MS/MS data are available via ProteomeXchange with identifier PXD008773. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: The data show that hnRNPCI 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 sihnRNPCI evidence that hnRNPCI could be an important player in the maternal-embryo communication established in the early stages of implantation.