| Research Background Placental implantation in human begins involves invasion of extravillous trophoblast into the uterine epithelium and underlying stromal cells. Trophoblast cells at human maternal-fetal interface show proliferation and invasiveness like tumor cells. However, in normal pregnancy, the invasiveness is strictly controlled, suggesting that the implantation process involves complex and synchronized molecular and cellular programs between the uterus and embryo, which is regulated by paracrine and autocrine factors. Trophoblast migration and invasion through the uterine wall is mediated by a series of molecular and cellular interactions controlled by the trophoblast and maternal microenvironment. Therefore, the related molecules involved in regulation of trophoblast invasion play an important role in the blastocyst implantation and pathological trophoblastic disease.Nometastatic gene 23-H1 (nm23-H1) is a wide-spectrum tumor metastasis suppressor gene. In the trophoblastic disease, the expression of nm23-H1 is closely related to the prognosis, as it can be used as a reference indicator to evaluate the progress. It is suggested that nm23-H1 may play a significant part in the invasion of trophoblasts. Therefore, further investigation to elucidate the mechanism of nm23-H1, and to find out how nm23-H1 is regulated might help us to understand the regulation of embryonic implantation and trophoblastic invasion.In the present study, we focus on the possible molecular mechanism of nm23-H1 on human trophoblast invasiveness during early pregnancy, and roles of the nm23-H1 in the cross-talking between trophoblast cells and decidual stromal cells, which contributes to trophoblast migration and ultimately to successful implantation. The identification of these pathways and molecule mechanisms could provide novel targets for the diagnosis and treatment of pathological pregnancies, enabling the translation of basic reseach discoveries into clinical applications. 1. The expression of nm23-H1 at human maternal-fetal interfaceMethods and Results We found by RT-PCR that all the deciduas and villi from the early pregnancy express moderately nm23-H1mRNA; while immunocytochemistry shows that the trophoblast cells and DSCs of the primary culture are all positively stained with anti-nm23-H1 antibody. The villi and decidua tissues from normal and spontaneous abortion were collected to detect the nm23-H1 expression. The RT-PCR results show that the villi and deciduas from normal early pregnancy appear a much lower nm23-H1 expression level than that of the miscarriage (P<0.01), and similar results have been demonstrated by Western blot and immunohistochemistry. Conclusion The nm23-H1 is expressed in villi and decidua, trophoblast cells and DSCs isolated from normal early pregnancy shows a much lower nm23-H1 level than that of the miscarriagea, which suggests that nm23-H1 may play an important role at human maternal-fetal interface in the first trimester pregnancy.2. Modulation of the pregnancy-associated hormone and proinfalmmatory molecule on nm23-H1 expression at human maternal-fetal interface in the first trimester pregnancyMethods and Results After isolation and culture of human trophoblast cells and DSC from the first trimester pregnancy, the primary cells were treated in vitro by the pregnancy-associated hormones, E2, progesterone and hCG, or proinflammatory cytokines, respectively. The nm23-H1 expression was determined by in cell western.It has been found that progesterone, orβ-hCG significantly down-regulates the expression of nm23-H1 in trophoblast cells. There is no detectable difference in expression level of nm23-H1 in the DSC culture alone after being treated by E2, progesterone, or low concentration of hCG; but high concentration of hCG treatment can down-regulate the expression of nm23-H1 in the DSC.Low concentration of LPS can down-regulate the expression of nm23-H1 both in trophoblast cells and DSCs. CsA can significantly inhibit the expression of nm23-H1 in the trophoblast cells, while CsA can not change the expression of nm23-H1 in DSCs alone.Conclusion It is concluded that both progesterone andβ-hCG can suppress the expression of nm23-H1 in trophoblast cells, which contributes to successful pregnancy. CsA can down-regulate nm23-H1 expression in human trophoblasts, which showes a noval mechanism by which CsA can improves pregnancy outcome.β-hCG in a high concentration can regulate the expression of nm23-H1 in DSCs, which suggests the trophoblast in trophoblastic diseases can suppress nm23-H1 expression in DSCs via secreting high concentration ofβ-hCG..3. The nm23-H1 inhibits invasiveness of human trophoblasts in the first-trimester prgnancyMethods and Results After isolation, purification and characterization of the first-trimester human trophoblast cells and DSC, a model of direct and indirect co-culture was set up. The expression of nm23-H1 in trophoblast cells and DSCs was interfered with siRNA, respectively. The effects of nm23-H1 on trophoblast invasion were investigated by Matrigel invasion, while the invasion-related molecules in the interfered cells were detected by in-cell western.The invasiveness of trophoblast cells is significantly enhanced in either direct or indirect co-cultures between trophoblast and DSC after the nm23-H1 is knock down in either trophoblasts or DSCs. The expression of titin increases significantly after the nm23-H1 is interfered in either trophoblasts or DSCs.Conclusion The results above suggest nm23-H1 interference may promote the expression of titin in both trophoblats and DSC, and in turn improves invasiveness of trophoblasts in coculture with DSCs.4. The nm23-H1 inhibits the invasiveness of human trophoblasts in the first trimester pregnancy by downregulating titin via MAPK/ERK1/2 signal pathwayMethods and Results After isolation, purification from the first-trimester pregnancy, human trophoblast cells and DSCs were interfered for nm23-H1, the key molecules involved in cell invasion were detected by in cell western to screen the possible signal pathway. It has been found that MAPK/ERK pathway plays an important role in the trophoblast invasiveness. Then we detected the protein level of P/T-Erk of the cells before and after the interference. It has been found that the activated P-Erk level increases obviously after nm23-H1 is knocked down. Thereafter, we set up co-culture models in vitro in combination of the interfered nm23-H1 with U0126, MAPK/ERK pathway inhibitor, and then the invasiveness of trophoblast cells was analyzed by Matrigel invasive assay, and the titin expression in the target cells was analyzed by in cell western, respectively. It has been found that the nm23-H1 knock down significantly promotes the invasion of human trophoblast cells and protein expression of titin in the cells; the inhibitor of MAPK/ERK1/2 signaling pathway, U0126, can significantly but not completely abolish the invasiveness increase. We investigated whether the enhanced titin was involved in the up-regulation of interfered nm23-H1 on invasion of trophoblast cells by statistical relationship analysis. It has been also found that the expression of titin is in positive relation to the invasiveness of the primary trophoblast cells.Conclusion These results above suggest nm23-H1 knock down improves the expression of titin in both human first-trimester trophoblasts and DSCs via MAKP pathway, and the up-regulated titin in turn promotes the invasiveness of trophoblasts in an autocrine or paracrine manner.In conclusion, nm23-H1 is involved in crosstalking at maternal-fetal interface: (1) suppressing the invasiveness of human first-trimester trophoblasts through titin accentuation via MAPK/ERK1/2 signal pathway; (2) the nm23-H1 expressed by DSC controls the over-invasion of trophoblast cells in decidua. The expression of nm23-H1 is regulated by pregnancy-associated hormones and CsA. Our results elucidate regulation mechanism of human trophoblast invasion, and provide a new idea for therapeutics of some pregnancy complications, such as miscarriage. |
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