Study On Serum Exosomal MiRNAs As A Diagnostic Marker For Early Pregnancy In Sows

The diagnosis of early pregnancy in sows is beneficial to shorten the empty pregnancy period of non-pregnant sows and improve the fecundity and production efficiency of sows.Therefore,it is very important to find and develop a new type of pregnancy biomarker.Exosome is a kind of extracellular vesicle containing various nucleic acids,proteins and lipids,which can be used as a communication factor for cell communication.The micro RNA carried in it is an endogenous small molecule non-coding RNA,which is widely present in eukaryotes and participates in the regulation of various cellular processes.A large number of studies have shown that exosomes and miRNAs are involved in a series of life activities such as embryonic growth and development,neuromuscular proliferation and differentiation,sexual maturity and immune response,etc.And play an important role in regulating the embryonic development of mammalian early pregnancy and the development of endometrium and intrauterine environment.This paper studied the differential expression of serum exosome miRNAs in early pregnant sows and its preliminary mechanism,and explored the possibility of exosome miRNAs as a diagnostic marker in production practice.It also provides reference materials for analyzing the relationship between the changes of embryo and uterus in early pregnancy and the changes of circulating body fluids,the role of miRNA in embryo development in early pregnancy and improving the reproductive performance of sows.Firstly,the exosomes in the serum of sows were isolated by ultracentrifugation and showed their related characteristics by Transmission Electron Microscope Analysis,Nanoparticle Tracking Analysis,Western blot analysis and nano-flow analysis.The miRNAs in the exosomes and their expression profiles in various periods were also extracted.The expression levels of miRNAs in the same period were quantitatively analyzed by RT-q PCR in independent populations,and reliable differential miRNAs were selected as biomarkers.Then target genes were predicted for the differential miRNAs,and a double luciferase reporter vector experiment was constructed to verify the differential miRNAs.Further explore the expression level of differential miRNAs in systemic tissues,and preliminary explore the role of differential miRNAs in early pregnancy.The specific research results are as follows:In this study,Large White pigs during the estrus period(9th day of false fertilization)and pregnancy period(9th,12 th,and 15 th day of synchronization fertilization)were used as the research objects.The exosomes in the serum of sows were isolated by ultracentrifugation and showed their related characteristics by transmission electron microscope analysis,nanoparticle tracking analysis,western blot analysis and nano-flow analysis.RNA-Seq was used to analyze the expression profile of miRNAs in each period,and the differentially expressed miRNAs were selected in large samples for further verification by RT-q PCR,and reliable differential miRNAs were screened as early pregnancy markers.Then the target genes of the selected miRNAs were predicted,their biological functions were analyzed,and the expression of miRNAs in various tissues was combined to explore their role in early pregnancy.The specific research results are as follows:(1)Exosomes with good morphology and structure were obtained from sows at various stages by ultracentrifugation.Compared with the method of separating exosomes with a kit,ultracentrifugation can obtain larger amounts and denser exosomes.At the same time,there was no significant difference in the amount of exosomes isolated during early pregnancy.(2)A high score of sequencing quality was obtained by sequencing the library of earlypregnancy sow serum exosome miRNA.A large number of reads contained various small RNAs including miRNA,and many novel miRNAs were also found in miRNA prediction.The changes of these small RNAs and miRNAs may be related to the developmental regulation of sow body and embryo in early pregnancy.(3)Difference analysis of miRNAs expression profiles revealed by sequencing,a total of 11 differential miRNAs were selected based on 15 days of pregnancy.In another independent population,RT-q PCR was used to verify these differences in miRNAs.Two miRNAs,ssc-miR-92b-3p and ssc-miR-17-5p was screened out,and could be used as biomarkers for early pregnancy diagnosis.(4)Analyze the difference of all miRNAs,and perform target gene prediction and functional analysis of all the miRNAs shown in the sequencing results to find that they are related to multiple biological functions.The regulatory functions of ssc-miR-92b-3p and sscmiR-17-5p,which were significantly differentially expressed,were mainly performed on target genes in early pregnancy.HAND1,HCRTR1,PFKM,BSG and ATF6 B are the main targeted genes that play a regulatory role in the early stage of embryo implantation and reproduction.In summary,exosomes are present in the sow serum.The difference between exosomes during pregnancy and estrus is mainly reflected in their contents.The differentially expressed miRNAs and their target genes play an important role in regulating early pregnancy.miRNAs carried by exosomes can be used as biomarkers for early pregnancy.