ANXA7 And PDIA3 Regulate Trophoblast Proliferation And Apoptosis In Preeclampsia

Objective: To investigate the expression of ANXA7 and PDIA3 in placental trophoblasts of preeclamptic pregnancies.To investigate the effects of ANXA7 and PDIA3 on human trophoblast apoptosis,proliferation.To explore their regulatory mechanisms on trophoblast cell.Methods: We collected human placental tissues from patients with PE and normal pregnant women.The expression levels of selective genes in placental trophoblasts of normal pregnant women and preeclamptic pregnancies were analyzed by real-time quantitative polymerase chain reaction(qRT-PCR).Western blot assay and immunohistochemistry to verify the expressions of ANXA7 and PDIA3.The ANXA7-knockdown and ANXA7-overexpressing HTR8/SVneo cells were utilized for studying the function of ANXA7 in trophoblasts.The proliferation and apoptosis levels of trophoblast were examined with Western blot assay,flow cytometry,Cell Counting Kit-8 assay,Ed U assay and immunofluorescence staining.The migration and invasion levels of trophoblast were detected by transwell assay.The downstream signaling pathway of ANXA7 regulating cell proliferation and apoptosis was verified by using molecular inhibitor and Western blot assay.We also applied Western blot assay and immunohistochemistry to detect the apoptosis related proteins BCL2 and BAX to verify that aberrant apoptotic placenta was present in preeclampsia compared with normotensive pregnancies.Western blot assay,Cell Counting Kit-8 assay,flow cytometry,and 5-ethynyl-2’-deoxyuridine(Ed U)staining were utilized for studying the function of PDIA3 in trophoblast.The downstream signaling pathway of PDIA3 regulating cell proliferation and apoptosis was verified by using RNA sequencing and Western blot analysis.We also applied Western blot assay and immunohistochemistry to detect the expression levels of MDM2 and p21 in preeclampsia patients.Resluts: ANXA7 expression was significantly lower in placentas from patients with PE compared with that from normal pregnant controls.The expression level of PDIA3 in preeclamptic trophoblasts was decreased compared with normal pregnancies.Knockdown of ANXA7 induced cell apoptosis and inhibited cell proliferation in HTR-8 by downregulating BCL2 protein levels.Knockdown of ANXA7 can also impair the function of migration and invasion of trophoblast.Overexpression of ANXA7 reduced apoptosis and promoted HTR8 proliferation.Further analyses showed that ANXA7 knockdown inhibited the activation of the JAK1/STAT3 pathway in HTR-8 cells.We further verified the decreased BCL2/BAX ratio in the villous trophoblasts of preeclampsia,which resulted in exaggerated apoptosis.We found that si RNA-mediated PDIA3 knockdown significantly promoted apoptosis and inhibited proliferation in the HTR-8 cell line,while overexpression of PDIA3 reversed these effects.Furthermore,knockdown of PDIA3 inhibited MDM2 protein expression in HTR-8 cells,concurrent with marked elevation of p53 and p21 expression.Conversely,overexpression of PDIA3 had the opposite effects.Our study revealed that MDM2 protein expression was down-regulated and p21 was increased in trophoblasts of women with PE compared to women with normotensive pregnancies.Conclusion: ANXA7 and PDIA3 were decreased in preeclamptic trophoblasts compared with normal pregnancies.The decreased expression of ANXA7 protein in preeclampsia patients may cause the inhibition of JAK1/STAT3/BCL2 signaling pathway.We also found that increased apoptosis occurs in placenta of patients with PE.Decreased PDIA3 induces trophoblast apoptosis and represses trophoblast proliferation through regulating the MDM2/p53/p21 pathway.