The Effect Of LncRNA ISG15-AS-mediated ISGylation On Embryo Implantation In Goats

The loss of embryos caused by implantation failure is an important reason for the low reproduction rate of animals.How to improve the success rate of implantation has become the main problem in the application of animal reproduction and assisted reproduction technology.Interferon-τ(IFN-τ),a specific pregnancy recognition signal secreted by the ruminant pregnancy,plays a key role in inhibiting luteal lysis and promoting embryo implantation by activating the JAK/STAT/ISGs pathway.ISG15,as a ubiquitin-like protein strongly induced by IFN-τ,mainly functions through the ISGylation modification system mediated by activating enzyme E1(UBE1L),binding enzyme E2(UBE2E)and ligase E3(HERC5).However,the function and mechanism of ISGylation during the establishment of uterine receptivity and embryo implantation need to be further studied.In recent years,the functions and regulatory mechanisms of long non-coding RNA(lnc RNA)in the field of reproduction have attracted widespread attention.In the previous work,we used Illumina deep sequencing technology to obtain differentially expressed lnc RNAs regulated by IFN-τin the goat endometrial epithelial cells,and found an significant upregulated lnc RNA(named ISG15-AS)which located in the antisense strand of ISG15.It is speculated that ISG15-AS plays an important role in goat embryo implantation.In this study,IHC and q RT-PCR were used to detect the localization and expression of the key proteins of ISGylation modification system in goat uterus tissues during early pregnancy.Meanwhile,the regulatory effect of IFN-τ on the ISGylation modification system was analyzed.The effect of ISGylation on the endometrial receptivity of goats was detected by ISG15 overexpression vector in the immortalized goat endometrial epithelial cell lines(g EECs).Furthermore,the key target proteins regulated by ISGylation during endometrial receptivity were screened by protein immunoprecipitation(IP)and mass spectrometry.The function of ISG15-AS in the goat endometrial receptivity though ISGylation modification system was studied by RNAscope,FISH and ASO interference technology.The following results are obtained through experiments:1.The key proteins ISG15,UBE1 L and UBE2 E of ISGylation modification system were mainly expressed in the luminal epithelial cells and glandular epithelial cells of the goat endometrium during early pregnancy.In the goat uterine tissues from 5 to 18 days of early pregnancy,the expression level of the ISGyaltion modification system and its target gene USP18 increased significantly.The m RNA levels of ISGyaltion modification system and USP18 were significantly upregulated after treatment with 20 ng/m L IFN-τ in g EECs.2.Overexpression of ISG15 could significantly increase the m RNA levels of VEGF and OAS2 in g EECs,and significantly reduce OPN,B2 M,IGFBP1,GRP and PTGS2 m RNA levels,but has no significant effect on the expression levels of E-cadherin,ITGB3 and PTGS1 m RNA.3.The results of IP/mass spectrometry and bioinformatics analysis showed that after IFN-τ treated g EECs,a total of 262 modified ISGylation target proteins were obtained,of which 41 differentially expression,mainly related to the gap junctions between cells,apoptosis and phagosome formation.4.ISG15-AS was mainly expressed in the luminal epithelium,glandular epithelium and superficial stromal cells of the goat endometrium during early pregnancy.On days 5-18 of pregnancy,the expression level of ISG15-AS were significantly increased with the process of early pregnancy.IFN-τ could significantly increase the expression level of ISG15-AS in g EECs;Knockdown of ISG15-AS significantly induced the expression levels of ISG15,UBE1 L,HERC5 and USP18 in g EECs.In summary,this study found that IFN-τ could regulate the process of goat embryo implantation by activating the ISGylation modification system.ISG15-AS could inhibited the expression of ISG15 and then affected the establishment of endometrial receptivity though ISGylation modification system.