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Tracking and capturing of bioorthogonal labelled RNA carried by extracellular vesicles during maternal–embryo communication

Es-haghi, M., Haling, A., Lättekivi, F., Tankov, S., James, V., Nafee, T., Kõks, S. and Fazeli, A. (2018) Tracking and capturing of bioorthogonal labelled RNA carried by extracellular vesicles during maternal–embryo communication. Journal of Extracellular Vesicles, 7 (Supp. 1). p. 203.

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Background: During implantation window, the uterine epithelium acquires a receptive phenotype and is being prepared for the initial blastocyst attachment. This unique phenomenon may stem from embryonic–maternal crosstalk utilizing an intricate language. Extracellular vesicles (EV) could be a logical mean for maternal–embryo communication. The current investigation was aimed at deciphering the main signals exchanged between the mother and the baby.

Methods: The 5-ethynyl uridine (EU)-labelled trophoblast spheroids were cultivated with an endometrial cell line in a non-contact co-culturesystem.ThetrophoblastEU-labelledRNAwastrackedandcaptured in endometrial cells. The transferred labelled RNA was affinity-precipitated and purified using biotin-azide click chemistry. Total RNA-sequencing was conducted with synthesized cDNA from captured labelled and non-EU labelled RNA (background) (n=4).Differential expression analysis of RNA-seq data was performed using edgeR and limma packages to identify the transferred transcripts using differential enrichment as a proxy. The Integrative Genomics Viewer was used to validate the coverage of differentially enriched transcripts. The results were confirmed by quantitative PCR (qPCR).To establish the route of candidate RNA transfer, EVs were isolated from co-culture media using size-exclusion chromatography. Total RNA was extracted from EVs, EU-labelled RNA was affinity-precipitated and the absolute copy number of putatively transferred RNA sequences was quantified.

Results: Differential enrichment analysis demonstrated that the majority of putatively transferred transcripts were non-coding RNAs derived from the mir99alet7c cluster (Chromosome 21: LINC00478). The presence of non-coding sequences from this chromosomal region in the RNA extracted from EVs was confirmed by qPCR. This suggests that these sequences are carried by throphoblast EVs.

Summary/Conclusion: In this study, we showed that biorthogonal RNA labelling chemistry can be used for the deciphering trophoblast–endometrial communications. These are the initial steps towards decoding the earliest stages of the mother–offspring language/crosstalk.

Item Type: Journal Article
Publisher: Taylor and Francis Open
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