STAT3 Signaling Pathway Is Involved In The Pathogenesis Of Early Spontaneous Abortion By Down Regulating CyclinD1 And VEGF

Objective To detect the expression levels of the STAT3 signaling pathway and its downstream genes such as Cyclin D1,VEGF,etc.,and the important vascular endothelial growth factor receptor protein VEGFR1 in the villi tissues and decidua tissues of normal women,patients with spontaneous abortion or recurrent abortion.To explore the pathogenesis of spontaneous abortion that STAT3 pathway may be involved.Methods From December 2017 to August 2018,we selected 80 patients undergoing painless abortion in the operation room of Obstetrics and Gynecology of the First Affiliated Hospital of Anhui Medical University.Among them,30 healthy women who voluntarily performed abortion due to social factors were in the control group,30 patients who with spontaneous abortion for early unexplained spontaneous abortion(EUSA)group,another 20 patients were defined as early unexplained recurrent spontaneous abortion(EURSA)group because they had two or more spontaneous abortions with the same sexual partner.All the three groups were performed painless artificial abortion in the early pregnancy(6-9 weeks gestation)and the villi and decidua of the three groups were collected.Hematoxylin eosin staining was used to observe the difference of villi in pathology among the three groups.The expression of phosphorylated STAT3(p-STAT3),Cyclin D1,VEGF and VEGFR1 were detected by immunohistochemistry,western blotting and real-time quantitative polymerase chain reaction.The human extravillous trophoblast cell line(HTR-8 / SVneo)was selected to establish a model of extravillous trophoblasts for cell experiments.Htr-8 /SVneo cells were treated at different concentrations of STAT3 signaling pathway inhibitor AG490(0,6.25,12.5,25,50,100 M)for 24 h,48 h and 72 h,respectively.We used an inverted optical microscope to photograph cell growth at different concentrations of inhibitors for 48 hours,then,western blot and q-PCR were used to detect the expression levels of p-STAT3,Cyclin D1 and VEGF in gradient inhibitor culture environment.CCK-8 kit was used to detect the proliferation of cells 48 hours after the action of gradient concentration inhibitors,and the apoptosis of villus trophoblast cells was analyzed by flow cytometry under the condition that different concentrations of drugs inhibited the STAT3 pathway.In addition,western blot was used to detect the expression of VEGFR1 in trophoblast cells under different degrees of STAT3 pathway inhibition,and to explore the changes in the ability of angiogenesis of trophoblast cells after STAT3 pathway inhibition.Results 1.Comparison of general conditions between the control group and the two experimental groups(EUSA group and eursa group)There was no significant difference in age,BMI,pregnancy,gravidity and time since gestation among the three groups(P > 0.05).2.Pathological differences of HE staining in three groups of villi Compared with the control group,the number of trophoblast cells decreased in EUSA group and EURSA group after HE staining,while some cells showed nuclear shrinkage and the number of apoptosis increased.3.Expression of Cyclin D1,VEGF and VEGFR1 in three groups of villi Immunohistochemical staining showed that the staining of Cyclin D1,VEGF and VEGFR1 in the normal control group was significantly stronger than that in EUSA group and EURSA group.Meanwhile,protein levels(western blot results)and m RNA levels(q-PCR results)showed that the decidua of Cyclin D1,VEGF and VEGFR1 in EUSA group and EURSA group were significantly lower than those in the control group(p < 0.05).4.Expression of Cyclin D1,VEGF and VEGFR1 in three decidual tissues Immunohistochemical staining,Western blot and q-PCR results showed that the expression levels of Cyclin D1,VEGF and VEGFR1 in the normal control group were significantly higher than those in EUSA group and EURSA group(p < 0.05).5.Expression of activated STAT3(p-STAT3)in three groups of villi and decidua Immunohistochemical staining showed that the specific staining of p-STAT3 in villus and decidua of normal control group was significantly stronger than that of EUSA group and EURSA group.When STAT3 in each group was approximately the same,the protein level of p-STAT3 decreased in EUSA group and EURSA group(chorion and decidua).6.Effect of inhibition of STAT3 pathway activation on trophoblast proliferation.The phosphorylation of STAT3 in HTR-8/SVneo cells cultured with gradient concentration inhibitors(0,6.25,12.5,25,50,100?M)AG490 was significantly inhibited(Western blot results).The growth of trophoblasts was observed under inverted fluorescence microscope with different concentrations of inhibitors.With the increase of inhibitor concentration,trophoblasts proliferation decreased and apoptosis increased.CCK-8 test and flow cytometry showed that inhibition of STAT3 pathway activation limited the proliferation of trophoblasts and increased apoptosis.7.As the activation of STAT3 was inhibited by gradient,Western blot results showed that the expression of Cyclin D1 and VEGF was decreased in a dose-dependent manner.8.Western blot showed that the expression of VEGFR1 decreased in a dose-dependent manner when STAT3 pathway was inhibited.Conclusion : The results of this study showed that STAT3 pathway in villus and decidua of patients with unexplained spontaneous abortion was significantly inhibited,and the expression of Cyclin D1 and VEGF,the downstream regulatory protein of STAT3,was decreased,which led to the growth restriction of trophoblast cells,the increase of apoptosis,and the decrease of VEGFR1,the important receptor of angiogenesis,which led to the damage of vascular endothelial growth,it may affect the growth and development of early embryos and cause miscarriage,which may provide some guidance for the clinical treatment of patients with spontaneous abortion for unknown reasons.