The Mechanism Research Of MiR-21 Regulating Apoptosis And Migration Of Porcine Endometrial Cells By Target PDCD4

Embryo implantation is a key link in mammalian reproduction.The expression of the internal genes and proteins in embryo and maternal are changed before and after embryo implantation,and this process is regulated by many factors.As a kind of endogenous regulatory factors,miRNAs play an important role in regulating embryonic processes.However,there are few reports on the effects and mechanisms of miRNAs on embryo implantation in pig.Through early research and analysis,it was found that miR-21 is highly expressed and differentially expressed in the endometrial tissues of pig embryo implantation stage,while the previous reports on miR-21 mostly focused on tumors.In order to explore the effects of miR-21 on porcine embryo implantation,we first examined the mRNA expression of miR-21 in endometrial tissues during the period of early pregnancy(days on 9,12 and 15)and on day12 of non-pregnancy in pigs,then we explored the miR-21 and target gene PDCD4 biological function of endometrial cell and embryo implantation by Annexin V-FITC flow assay,Transwell chamber assay,mouse uterine horn injection,qRT-PCR and Western Blot.The research results lay a foundation for understanding the molecular mechanism of pig endometrial cells on the implantation of pig embryos.what's more,This is of great significance to the improvement of the economic benefits of the pig industry.The main results and conclusions are as follows:1.qRT-PCR was used to detect the mRNA expression of miR-21 in endometrial tissues during the period of early pregnancy(days on 9,12 and 15)and on day12 of non-pregnancy in pigs.The results showed that miR-21 had the highest relative expression on day 12 in the endometrium.There are significant differences between the 12 th day of pregnancy and the 9th day of pregnancy and the 12 th of non-pregnancy.2.Annexin V-FITC flow cyctometry results showed that inhibiting the expression of miR-21 can promote the apoptosis of pig endometrial cells.Transwell chamber assays showed that inhibiting the expression of miR-21 can reduces the migration ability of pig endometrial cells.3.Bioinformatics predicted that miR-21 can target the PDCD gene.Dual-luciferase reporter assay showed that miR-21 can bind to the 3' untranslated regions(3'UTR)of PDCD4 gene;qRT-PCR and Western blotting further identified that miR-21 can negatively regulate the expression of PDCD4 at the level of translation.4.Annexin V-FITC and Transwell chamber assays showed that inhibition PDCD4(transfection of PDCD4 siRNA)can inhibit apoptosis of endometrial cells and promote cells migration.Supplementary exogenous PDCD4 siRNA may be able to weaken promote endometrial cells apoptosis,which is due to inhibition miR-21 and reply the inhibitory effect of miR-21 inhibitor on the migration of pig endometrial cells.5.Through the qRT-PCR detection showed that inhibition of miR-21 expression can significantly down-regulated the mRNA expression of TGF-? gene.Knockdown PDCD4 gene would decrease the mRNA expression of TGF-? gene in porcine endometrial cells.6.The results of in vivo uterine horn injection test showed that the number of mouse embryos was significantly reduced after injection of the miR-21 interference fragment.In summary,after down-regulating miR-21,it directly targets PDCD4 gene to promote apoptosis of pig endometrial cells,inhibit cell migration,and inhibit the expression of embryonic accessory marker gene TGF-?,down-regulation of miR-21 will directly affect the embryo implantation of mice,which is not conducive to embryo attachment.It is speculated that miR-21 may play an important role in early embryo implantation of pigs.