| Placental trophoblast cells play an important role in embryo implantation process during the early pregnancy. Moderate function of trophoblast cells, including proliferation, apoptosis, invasion and migration, is the key to establish, maintain and timely termination of normal pregnancy. Excessive function of trophoblast cells may lead to trophoblastic tumors (such as hydatidiform mole, choriocarcinoma etc.); Weak function of trophoblast cells can lead to pregnancy failure, such as spontaneous abortion, premature birth, and pathological pregnancy, such as preeclampsia and intrauterine growth restriction. The molecular mechanism of dysfunction of trophoblast cells has been extensively studied. However, which is the key targets and through which pathway, the protein level changes resulting in the biological behavior changes of trophoblast cells is still not clear.S100P is a member of the S100 family of calcium-binding proteins, which was first detected in human placental tissue, indicating the possible relationship between S100P and pregnancy. However, there are, to date, no reports describing the expression and distribution of S100P in human placenta during the entire pregnancy and certainly not the cell type involved and the relationship between S100P and spontaneous abortion, gestational trophoblastic tumor also remains unknown.We first examined the S100P in placental tissue in different periods of entire pregnancy, in villi of spontaneous abortion of early pregnancy and gestational trophoblastic tumor; secondly, to study the effects of S100P on the biological function of trophoblast cells and possible molecular mechanisms through in vitro and in vivo experiment. The purpose of this study was to investigate the relationship between the expression of S100P and the function of placental trophoblast cells.PART â… The Expression of S100P in Platental TissueObjective:To compare the S100P expression in placental trophoblast cells from different stage of pregnancy and gestational trophoblastic tumors.Materials and Methods:In this experimental study, the mRNA expression levels of S100P in placenta tissues were detected by reverse-transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. The protein expression levels were detected by western blot, and the localization of S100P was measured by immunohistochemical staining.12 first-trimester placental tissue,10 second-trimester placental tissue, and 12 term placentas, were collected to detect the S100P expression in different stage of normal pregnancy.16 chorionic villi of first trimester spontaneous abortion were collected to detect whether the S100P expression is changed, compared with group of villi of normal pregnancy.23 cases of gestational trophoblastic tumor tissue were collected, including 10 cases of hydatidiform mole,3 cases of invasive hydatidiform mole and 10 cases of chorionic carcinoma, of which 5 cases also has primary tumor and pulmonary metastatic tumor. The S100P protein expression were detected by immunohistochemical staining.Results:(1) High protein and mRNA expression of S100P could be detected in placenta during pregnancy, with higher levels in first-trimester. Immunohistochemical staining revealed that S100P protein was strongly expressed in syncytiotrophoblasts, and moderate expression was detected in villous cytotrophoblasts and cytotrophoblast columns. The S100P protein was localized to both cytoplasm and nuclei in syncytiotrophoblasts, while it only existed in the cytoplasm of cytotrophoblasts. In secone-trimester and term placenta tissue, strong S100P immunoreactivity was still localized at the cytoplasm and nuclei of syncytiotrophoblasts.(2) The protein and mRNA expression level of S100P significantly decreased in early spontaneous abortion villi, compared with group of villi from normal pregnancy.(3) Immunohistochemical analysis results showed that S100P protein stainig is strong positive in the syncytiotrophoblasts in some patients of 10 cases of hydatidiform mole (3/10), while moderate or strong positive staining in cytotrophoblasts; and in 3 cases of invasive hydatidiform mole (3/3),6 cases of choriocarcinoma primary foci (6/10) and 5 cases of pulmonary metastasis tumor tissue (5/5), the S100P protein was strongly positive in cytotrophoblasts.Conclusion:S100P was strongly detected in human placenta during pregnancy. The specific expression and distribution of S100P in human placenta throughout gestation suggested that S100P function might vary with its location in the placenta. The expression of S100P in chorionic villi of spontaneous abortion from early pregnancy was decreased and the expression of S100P in gestational trophoblastic tumor was elevated, which suggested that the expression of S100P might be closely related to the function of placental trophoblast cells.PART â…¡:The Effect of S100P on the Biological Function of Trophoblast Cells and its Molecular MechanismObjective:To investigate the effect of S100P on the biological behavior of trophoblast cells and its possible molecular mechanism.Materials and methods:JAR and JEG-3 trophoblast cell lines were cultured in vitro. The constructed eukaryotic expression vectors and small interfering RNA were transfected into JAR cells and JEG-3 cells respectively. Different groups of trophoblast cells were divided. The behavior of trophoblast cells were detected using XTT cell growth assay, flow cytometry assay, plate clone formation assay and transwell cell migration assay, to analyze the effect of S100P on the ability of growth, apoptosis, clone formation and migration of trophoblast cells. The effect of S100P on the ability of JAR and JEG-3 in vivo and the ability of metastasis of the tumor cells were observed by subcutaneous injection into nude mice and the tail vein injection. To further explore the mechanism of action of S100P, we used western-blot to detect the role of P38 signaling pathway related molecules in JAR cells.Results:(1) We constructed the S100P eukaryotic expression vector and two pairs of S100P small interference RNA (siRNA1, siRNA2), which were used to transfect the trophoblast cell lines JAR and JEG-3. S100P over-expression promoted the growth, colony formation and migration capacity of trophoblast cells in vitro, and inhibited cell apoptosis, when compared with the control group (P<0.05). In contrast, S100P down-regulation by transfection of siRNA significantly reduced the colony formation and migration capacity of trophoblast cells, compared with the control group(P< 0.05).(2) We then examined whether S100P over-expression can promote tumor formation and trophoblast cell metastasis in nude mice. The results showed that, the trophoblastic tumor weight was significantly greater in S100P overexpression group, than that of the control group and the difference is statistically significant (P<0.05). At the same time, the metastasis of the tumor cells showed that S100P over-expression also enhanced the trophoblast cell metastasis in nude mice, compared with the control group (P<0.05).(3) The results of experiment study for molecular mechanism demonstrated that up-regulation of S100P in JAR cells increased the expression of phosphorylated P38 (P-P38) mitogen-activated protein kinase(MAPK) and P-ERK MAPK. However, the effects of S100P on JAR cells proliferation were prevented by P38 inhibitor-SB203580, but not by ERK inhibitor-PD98059.Conclusion:S100P can promote the proliferation and migration of trophoblast cells in vitro and tumorigenesis and metastasis in vivo, and S100P may promote the proliferation of trophoblast cells through modulating the P38 MAPK signaling pathway. |
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