The Mechanism Research Of ANXA8 Regulating The Proliferation Of Porcine Endometrial Cells

Embryo implantation is one of the most sensitive period for early embryonic losses of porcine,which is regulated by some factors.However,reports of factors associated with embryo implantation are fewer.Previous of RNA sequencing analyses and iTRAQ sequencing analyses in our laboratory have shown that differential expression of ANXA8 is founded on endometrium during the pre-implantation phases in pig.ANXA8 gene is a member of the Annexin family,it encodes an anticoagulant protein which is involved in the blood coagulation cascade and acts as an indirect inhibitor of the thromboplastin-specific complex.Whereas,the biological function of ANXA8 in porcine endometrial cells still remains largely unknown.In order to make sure the biological function of ANXA8 in porcine endometrial cells,the mRNA expression of ANXA8 in porcine endometrium tissues was detected during early pregnancy.Then,the study confirmed the regulating effect of ANXA8 on porcine endometrial cells by using immunofluorescence,real-time RT-PCR,cell cycle,western blot,before gene transfering of pcDNA3.1(+)-ANXA8 plasmid and siANXA8 into porcine endometrial cell.Intrauterine injection of mice analysis was used to confirm the effect of ANXA8 on embryo implantation in mice.The main results:1.Real-time RT-PCR analysis was used to determine the mRNA expression of ANXA8 in the endometrium on days 9-15 and 18 of pregnancy in pigs.It was found that the mRNA expression of ANXA8 in the endometrium was the highest level on day 12.And immunofluorescence analysis was performed to identify the localization of ANXA8 protein expression in porcine endometrial cells.The results showed that the protein was expressed in the cytoplasm of porcine endometrial cells.2.Real-time RT-PCR analysis was used to determine that ANXA8 up-regulated the mRNA expression of LIF and EGF in porcine endometrial cells.3.We performed cell cycle analysis to determine ANXA8 promoted the porcine endometrial cells had an increase in the S phase.Immunofluorescence analysis and western blotting analysis were performed to identify that overexpression ANXA8 increased the abundance of PCNA(a proliferation marker).4.Overexpression ANXA8 can promote the expression levels of p-AKT of porcine endometrial cells through the AKT signaling pathway by western blot analysis.Immunofluorescence analysis and western blot were used to determine ANXA8 promotes proliferation of endometrial cells through the signaling pathway of AKT.In porcine endometrial cells with both ANXA8 overexpression and the addition of perifosine,the expression of PCNA protein was not significantly different from that in the control groups,and was obviously lower than that in the cells with ANXA8 overexpression by immunofluorescence analysis;The western blot results indicated that perifosine has no effect on the expression levels of ANXA8 protein in porcine endometrial cells.We determined that overexpression ANXA8 up-regulates the mRNA expression of mTOR(AKT downstream gene)in endometrial cells by real-time RT-PCR.5.Intrauterine injection of mice was used to determine whether the expression of ANXA8 affects embryo implantation in vivo,the results indicated that knockdown of ANXA8 would decrease the implantation sites in mice.6.Knockdown ANXA8 would decrease the mRNA expression of ANXA4(ANXA8 homologous gene)in porcine endometrial cells by real-time RT-PCR.The aforementioned main results showed that ANXA8 could promote the proliferation of porcine endometrial cells via the AKT signaling pathway.In addition,ANXA8 affects the mRNA expression of LIF and EGF in porcine endometrial cells,LIF and EGF are the embryo implantation marker genes.Knockdown ANXA8 also affects the embryo implantation sites in mice.