Sequencing Of Small RNA In Extracellular Vesicles Of Uterine Fluids During Porcine Embryo Implantation And The Function Of MiR-92b-3p

The success of embryo implantation is a key factor in determining the litter size in pigs.The extracellular vesicles(EVs)in the uterine fluids could influence the embryo implantation by mediating the communication between the endometrium and the embryo.The EVs in uterine flushing fluids(UFs-EVs)on days 10,13 and 18 of pregnancy(D10,D13 and D18)were used as the research object,and then we extracted their small RNA for sequencing and bioinformatics analysis.In order to understand the effect of UFs-EVs during embryo implantation on endometrial epithelial cells(EECs)and embryonic trophoblast cells(PTr2 cells),The UFs-EVs on the D13 were added to the culture medium of EECs and PTr2 cells,and then we perform transcriptome sequencing analysis on the two groups of cells.Finally,we screened out a differentially expressed(DE)gene(mi R-92b-3p),and studied the functional effects and regulatory mechanisms of the mi R-92b-3p in the EVs derived from EECs(EVs-mi R-92b-3p)on PTr2 cells.The main research results are as follows:(1)We observed that multivesicular bodies enclosing multiple cup-shaped vesicles in the porcine endometrial tissue during the embryo implantation period by transmission electron microscopy.We also found that there are small spherical vesicles with a diameter of 100-400 nm on the surface of embryos during embryo implantation period by scanning electron microscopy.The results of immunohistochemistry showed that the protein CD9,which is related to the secretion and uptake of extracellular vesicles,expressed in endometrial luminal epithelial cells on the D10,D13 and D18 during embryo implantation period and in the embryonic trophoblast cells on the D18.(2)In this study,UFs-EVs from sows on the D10,D13 and D18 were separated.Western blot analysis revealed that the UFs-EVs fraction was positive for EVs-related protein markers(CD63,CD9 and HSP70)and negative for calnexin.The cup-shaped UFsEVs were observed by transmission electron microscope.In addition,separated UFs-EVs were subjected to nanoparticle tracking analysis,revealing that their sizes ranged from 40 to 160 nm.Then a comprehensive analysis of the small RNA in these UFs-EVs expression profiles was carried out.A total of 152 known micro RNAs(mi RNAs),43 new mi RNAs,6248 known piwi-interacting RNAs(pi RNAs)and 110 new pi RNAs were identified.RTq RCR results showed that ssc-let-7f-5p,ssc-let-7i-5p and ssc-let-7g were differentially expressed during the three stages.Bioinformatics analysis showed that the DE mi RNAs in the three comparison groups(D10 vs D13,D13 vs D18 and D10 vs D18)were related to important processes and pathways related to immunity,endometrial receptivity and embryonic development.(3)The PKH67 labeling UFs-EVs on D13 were co-cultured with EECs and PTr2 cells for 12 h and 24 h.We found that fluorescently labeled UFs-EVs on the D13 could be taken up by EECs and PTr2 cells via laser confocal scanning microscopy.(4)In this study,transcriptome sequencing was performed on EECs and PTr2 cells treated with UFs-EVs on the D13.After treatment of UFs-EVs,there were 1793 and 6279 genes differentially expressed in the EECs and PTr2 cells,respectively.By the RT-q PCR,we found that ID2,ITGA5,CXCL10,CXCL11 were DE genes in the comparison group of the two cells.Bioinformatics analysis showed that the DE genes in EECs and PTr2 cells after treatment are involved in immune regulation,cell migration,cell adhesion,and the secretion and uptake of EVs.(5)EVs were isolated from the EECs culture medium,and the EVs released by EECs were identified by transmission electron microscopy,western blot and nanoparticle tracking analysis.The CM-Dil labeling EVs derived from EECs and co-culture with PTr2 cells.Through laser confocal scanning microscopy,we found that fluorescently labeled EVs could be taken up by PTr2 cells.(6)Through the Transwell co-culture system,it was found that FAM-labeled mi R-92b-3p transfected EEC can deliver mi R-92b-3p to PTr2 cells by EVs,and the EVs-mi R-92b-3p can be taken up by PTr2 cells and enhance the expression of mi R-92b-3p in PTr2 cells.Both EVs-mi R-92b-3p and mi R-92b-3p can significantly promote the proliferation,migration and adhesion of PTr2 cells.Dual-luciferase analysis found that mi R-92b-3p can target TSC1 and DKK3,and mi R-92b-3p can inhibit the expression of m RNA and protein of TSC1 and DKK3.Function recovery experiments show that the 3’UTR vectors of TSC1 and DKK3 can recover the effects of mi R-92b-3p on the function of PTr2 cells.Furthermore,loss of function of mi R-92b-3p in vivo resulted in a decreased number of implanted mouse embryos.