The Research Of TET1 Regulated MMPs And TIMPs On Goat Trophoblast Cells

Placental trophoblast cells are the cells that appear in early pregnancy and are the main components of the placenta in early pregnancy.They have the functions of migration,invasion and secretion,and participate in multiple physiological processes such as embryo implantation and blood vessel formation.In recent years,a large number of studies have focused on the role of epigenetics in trophoblast cells.TET1(ten-eleven translocation1),as an important enzyme molecule in the active demethylation process,is involved in the regulation of multiple physiological functions in trophoblast cells.Migration and invasion are important functions to ensure embryo attachment,depending on the normal expression of matrix metalloproteinase(MMPs)and tissue inhibitor of metalloproteinase(TIMPs),but currently,there are few reports about such molecules in the placental trophoblast of goats.It is not clear whether TET1 can regulate the migration and invasion function by regulating the expression of such molecules.Therefore,this study designed in vivo and cell experiments to clarify the expression of TET1,MMPs,and TIMPs in the trophoblast of early goat pregnancy,and explored the effect of TET1on the migration and invasion of goat trophoblast cells.In this study,15 young black goats with healthy and empty sheep from Southwest University farms were selected to be induced estrus with pessary,and the trophoblasts and fetuses were taken until 20,25,30,45 and 60 days of pregnancy.The expression changes of TET family,MMP family and TIMP family members in trophoblasts were explored at different stages of early pregnancy.Then,shTET1 lentivirus was prepared,and the immortalized goat trophoblast cells(GTCs)were used as the research object.In this study,we used real-time fluorescent quantitation PCR,immunohistochemistry,immunofluorescence,Weestern Blotting,Transwell chamber migration and invasion assay to determine the effect on migration and invasion of GTCs by TET1.The main findings are as followed:1.The expression of TET1 in the trophoblasts at the25 d was significantly higher than that at 20 d,25 d,30 d,45 d and 60 d of pregnancy(P<0.05),and that at 45 d was significantly lower than 20 d,30 d,and 60 d(P<0.01).Compared with other members of the TET family,TET1 expression was significantly higher than TET3 at 20 d of gestation(P<0.05),significantly higher than TET2(P<0.01),and significantly lower than TET3 at45 d of gestation(P<0.01).TET1 protein was expressed positively in the nucleus and cytoplasm of placental trophoblast,and it was strongly expressed at 20 d,45 d and 60 d of pregnancy.At the same time,TET1 expressed in endometrial cells weakly in the placenta at 45 d and 60 d of gestation.2.ShTET1 lentivirus was prepared by 293T cells,40μL of virus protein with a concentration of 3.98×10~8 IU/mL was added to GTCs,cultured for 48 h,and then expressed by green fluorescence after 48 h of 12 mg/mL puromycin.GTCs,and TET1expression was significantly lower than Control group and shControl group(P<0.05).3.When TET1 expression was inhibited,the migration ability of GTCs was significantly decreased(P<0.05),and the invasion ability level was significantly lower than that of Control group and shControl group(P<0.01).There was no significant change in cell proliferation ability of GTCs.4.After knocking down TET1,the expression of MMP2 was significantly decreased(P<0.05).The expression of MMP9 was significantly lower than that of Control group and shControl group(P<0.01).The expression of TIMP1 was significantly lower than that of Control group(P<0.01).The expression of TIMP2 was significantly lower than that of the Control group and the shControl group(P<0.05).5.In the early stage of goat pregnancy,except for 20 d and 25 d,the expression of MMP9 was higher than that of MMP2,and the difference was significant at 30 d and 60d(P<0.05).The difference was extremely significant at 45 d(P<0.01).Moreover,the expression pattern of MMP9 was lower before 30 d,and then gradually increased.The expression of TIMP1 was higher than TIMP2 except for 20 days,and there was a significant difference at 30 d and 45 d(P<0.05).The expression trends of TIMP1 and TIMP2 were basically the same,which was lower at 20 d,and highest at 25 d,and then gradually decreased.6.MMP9 mainly showed positive expression on cytoplasm and cell membrane,and strong positive expression on 45 d and 60 d.At the same time,in the placenta of 45 d and60 d,MMP9 and TET1 were weakly expressed in uterine epithelial cells,and there was a certain similarity between the distribution and expression of the two.Conclusion:1.In the early gestational layer of goats,TET1 was highly expressed in early TET family members,and TET3 was highly expressed at 45 d,indicating that the two may alternately play a regulatory role.2.After the low expression of TET1,the migration and invasion ability of GTCs decreased,but the cell proliferation ability did not change significantly,indicating that the low expression of TET1 inhibited the migration and invasion of GTCs,but did not affect the proliferation ability of GTCs.3.After low expression of TET1,its inhibitory effect on MMP9 and TIMP1 gene expression was higher than that of MMP2 and TIMP2.4、MMP9 and TIMP1 may play a major role in migration and invasion.Also,the expression and localization of MMP9 in placental trophoblasts in early pregnancy were similar to those of TET1,suggesting that TET1 may affect the migration and invasion of placental trophoblast cells mainly through MMP9 and TIMP1.