Regulation Of ST6GAL1 Mediated α2,6-sialic Acid Modification On Proliferation Of Endometrial Epithelial Cells During Implantation In Pigs

To improve the ability of pig molecular breeding technology and germplasm innovation is the key problem that needs to be solved urgently in China’s pig industry.The litter size is the most direct measure of pig reproductive efficiency and plays a very important role in pig genetic improvement.Embryonic mortality is a key factor limiting the realization of litter size potential,and 30% of embryo death occurs in the stage of pig embryo implantation.Therefore,the study on the regulation mechanism of pig embryo implantation will help to analyze the genetic basis of pig litter size.Protein glycosylation modification is one of the main posttranslational modification,but because of the diversity of glycan structures,the current of protein glycosylation modified role in pig embryos with plant rarely reported.Sialic acid is a monosaccharide consisting of nine carbon atoms,often located at the end of the glycan structure to form the sialic acid modification of proteins.Our previous study found that:(1)α2,6-sialic acid structure was expressed in the endometrium cells of porcine embryos during implantation,and the expression pattern was different before and after implantation,only expressed in the endometrium during implantation(15 days of gestation).(2)The α2,6-sialic acid transferase gene ST6GAL1 is a key gene that mediates α2,6-sialic acid modification of proteins in endometrial epithelial cells.Therefore,this study first detected the proliferation ability of porcine endometrial epithelial cells at the implantation stage(15 days of gestation),which was consistent with the expression pattern of ST6GAL1 and α2,6-sialic acid structures in porcine endometrium found in our previous study.On this basis,we studied the effect of ST6GAL1 mediated α2,6-sialic acid modification on the endometrium regulation of cell proliferation.The main results were as follows:1,In this study,immunofluorescence staining was performed on pig endometrial tissues during early gestation,and it was found that the expression of cell proliferation marker protein KI67 was high in endometrium at 15 days of gestation,but only sporadic at 12 days of gestation.Meanwhile,integrin-matrix adhesion marker protein integrin-β1 was highly expressed at 12 days of gestation but low at 15 days of gestation in the endometrium,indicating proliferation of porcine endometrium epithelial cells at embryonic implantation(15 days of gestation).2,The α2,6-sialic acid structure is involved in the regulation of cell proliferation.In Ishikawa cells,the α2,6-sialic acid structure on the cell surface was covered with SNA lectin,and then the cell proliferation was detected before and after the covering.The results showed that the presence of α2,6-sialic acid structure could promote cell proliferation.3,The formation of α,2,6-sialic acid structure mediated by ST6GAL1 can promote the proliferation of cells.(1)The proliferation ability of cells was detected by RNA interference technique,and it was found that the proliferation ability of cells was significantly reduced by inhibiting the expression of ST6GAL1 gene(P <0.01);(2)Flow cytometry was used to detect the cell cycle of Ishikawa cells transfected with siRNA/NC.The results showed that the G1 phase of cell cycle arrest occurred in Ishikawa cells after the expression of ST6GAL1 gene was inhibited.(3)Hoechst staining was used to analyze the apoptosis of Ishikawa cells transfected with siRNA/NC,and it was found that Ishikawa cells showed apoptotic characteristics(nuclear hyper-chromaticity and nuclear fragmentation)after the inhibition of ST6GAL1 gene,indicating that the inhibition of the expression of ST6GAL1 gene would promote the apoptosis of Ishikawa cells.4,The ST6GAL1 gene mediated α2,6-sialacidification of GRP78 affects the proliferation of human endometrial cancer cells by regulating the endoplasmic reticulum stress pathway.(1)Ishikawa cells transfected with siRNA/NC were treated by immunoprecipitation with SNA lectin,and then analyzed the proteins by LC-MS/MS proteome sequencing.177 and 152 α2,6-sialic acid modified proteins were identified after siRNA/NC transfection,respectively,30% of these proteins are associated with endoplasmic reticulum stress.(2)Western blot analysis showed that the ER stress marker GRP78 was modified by α2,6-sialic acid.Further Western blot analysis of its downstream regulatory proteins showed that GRP78 modified by α,2,6-sialic acid could affect the proliferation of Ishikawa cells through regulating the PERK pathway.In conclusion,this study found that ST6GAL1 gene may affect the proliferation of endometrial epithelial cells through regulating the endoplasmic reticulum stress pathway by mediating the α2,6-sialacidification modification of GRP78.