The Function And Molecular Mechanism Of The Wnt/β-catenin Pathyway In RAS Cytotrophoblasts Cells

ObjectiveAbout 60-70 percent of the patients with RSA still can not find the aetiology of recurrent abortion.It’s very difficult for ctreatment. To date, numerous signalling factors and pathways have been found to be important for a successful early pregnancy. The Wnt/β-catenin signalling pathway is one of a evolutionarily conserved signal transduction pathways and plays important roles in cell proliferation, fate specification, polarity, and migration. Recent studies have found that many Wnt ligands and Wnt signalling related genes are dynamically expressed in the villous trophoblast epithelium and the uterine stroma during the process of trophoblast invasion and uterine decidualization. Whereas the precise molecular basis of reciprocal interactions between the mother and foetus during early pregnancy of RSA patients still remains largely unknown. Despite previous studies showed that DKK1 incresased in unexplained recurrent spontaneous miscarriage (URSM) patients and may be a valuable biomarker of URSM the association between abnormal expression of Wnt/β-catenin signalling and RSA remains unknown.To study the molecular mechanisms underlying Wnt/β-catenin signaling pathway in the progression of early pregnancy through investigating the expression of β-catenin and Dickkof-1 in first trimester villi and decidua of recurrent spontaneous abortion patients.MethodsVillous and decidual tissues were collected from 40 person (20 patients with recurrent spontaneous abortion and 20 patients with normal, early pregnancy). All participants enrolled were outpatients at the Department of Obstetrics and Gynecology, Yantai Yuhuangding Hospital, Shandong province. Twenty patients who had experienced at least two consecutive first-trimester losses (8.6±1.4 weeks of gestation) of unexplained aetiology, with an age (mean±SD) of 30.8±3.60 years age range,26-36 years) were recruited as participants. For a relevant control group, we selected 20 healthy women undergoing elective teration of apparently normal pregnancies. Western blots were used to measure the protein levels of β-catenin in villi and decidua, and the localization of P-catenin was investigated by immunohistochemistry. Quantitative real-time RT-PCR was used to quantify the mRNA levels of P-catenin and Dickkof-1 in villi and decidua, respectively.ResultsOur results indicated that β-catenin was expressed predoantly in plasma membranes of the villous cytotrophoblasts and glandular epithelium. What’s more, its epxression significantly decreased at both mRNA and protein levels, whereas the mRNA levels of Dickkof-1 significantly increased in villi and decidua of recurrent spontaneous abortion group compared with normal control group.ConclusionsThe cell-specific distribution of P-catenin in first trimester mononucleate villous cytotrophoblasts suggested that β-catenin mainly plays roles in trophoblast proliferation at early pregnancy. Immunohistochemistry showed that P-catenin was mainly localized in glands epithelium of the decidual tissues. It is well known that decidual glands may play more active roles in trophoblast invasion and placental and embryonic growth at early stage of pregnancy though secreting growth factors, indicating that decreased expression of β-catenin in decidual tissues may be associated with the pathogenesis of RSA. Our results demonstrated that P-catenin was mainly localized in the first trimester mononucleate villous cytotrophoblasts of the normal and RSA groups, Moreover, a nuclear localization of β-catenin was also dectected in both the RSA and control groups, which suggested an proliferative status of villous cytotrophoblast. In the present study, we detected an abnomal expression of Wnt/β-catenin signalling in RSA, p-catenin significantly decreased, whereas the DKK1 significantly increased in villi and decidua of RSA compared to normal controls, which suggested a inhibition of Wnt/p-catenin signalling in RSA. It implied that down-regulated Wnt/β-catenin signalling in RSA may associated with the inhibition of the trophoblast proliferation and the uterine decidualization, which often occurred in early spontaneous abortion. We therefore speculated that down-regulated Wnt/β-catenin signaling pathway might be associated with the process of the pathogenesis of recurrent spontaneous abortion.BackgroundWingless ligands, a family of secreted proteins, are critically involved in organ development and tissue homeostasis by ensuring balanced rates of stem cell proliferation, cell death and differentiation. WNT signaling components also play crucial roles in trophoblast development, proliferation, migration, infusion, placental function, undergo changes in endometrial and gestational diseases.14 WNTligands and 8 Fzd receptors were found to be expressed in total first trimester placenta. Most of these mRNAs were detectable in the villous trophoblast epithelium and endometrium. Moreover, cell-specific distribution of WNT ligands could also be observed. WNT1, WNT7b, WNT10a, and WNT10b were down regulated from first trimester to term suggesting roles in trophoblasts of early pregnancy. WNT1 abundantly expressed in first trimester CTBs and EVTs. WNT2 and WNT3 moderately expressed in CTBs and EVTof first trimester. WNT7b、WNT9b were highly expressed in first trimester villous cytotrophoblasts but expression decreased during EVT formation suggesting different WNT pathways may operate during human trophoblast development. WNT1, WNT2 and WNT3 could play a important role in early trophoblast development, cell proliferation, differentiation, migration and infusion.WNT-2, WNT-4, WNT-5a, WNT-7a, LRP-6, and β-catenin were equally expressed in proliferative and secretory phase endometrium. However, Dkk-1 was highly expressed by stromal cells in the secretory phase of the cycle, with no or little expression in the proliferative phase. WNT-3 was significantly higher in the proliferative than in the secretory phase. β-catenin was found to be expressed in glandular, as well as stromal cells, and is abundant during both the proliferative and secretory phases of endometrial cycle. Which suggest that the expression of Wnt/β-catenin and DKK1 in endometrium play an important role in trophoblast migration and infusion. Moreover, the endometrial WNT ligands, WNT2, WNT4, WNT5a, WNT7a, WNT8b, and WNT3, the latter being regulated during the menstrual cycle, could affect trophoblast function in a paracrine mannerObjectiveOur preliary work research has proved that the Wnt/β-catenin signaling pathway in the patients with RSA is suppressed. But it was not clearly that which key WNT ligands affect villous trophoblast cell proliferation and reducing the invasion. The project is proposed on the basis of previous work, using all kinds of cell biology and molecular biology methods to screen difference WNT ligands in the trphoblast and decidual of normal and RSA to revealing the mechanism of Wnt/p-catenin signaling pathway how to affect the cell proliferation, migration, from a new perspective to explore the pathogenesis and treatment of unexplained recurrent abortion.MethodsVillous and decidual tissues were collected from 40 person (20 patients with recurrent spontaneous abortion and 20 patients with normal, early pregnancy). All participants enrolled were outpatients at the Department of Obstetrics and Gynecology, Yantai Yuhuangding Hospital, Shandong province. Twenty patients who had experienced at least two consecutive first-trimester losses (8.6±1.4 weeks of gestation) of unexplained aetiology, with an age (mean±SD) of 30.8±3.60 years age range,26-36 years) were recruited as participants. For a relevant control group, we selected 20 healthy women undergoing elective ter分�'�ation of apparently normal pregnancies. Quantitative real-time RT-PCR was used to quantify the mRNA levels of WNT1、WNT 2、WNT 2b, WNT 3、 WNT 3a, WNT 4, WNT 9b, WNT10A and WNT 10B respectively. Western blots were used to measure the protein levels of MMP-2, MMP-9 and CyclinD1, MTT and Cell scratch experiment to evaluate the proliferation and the ability of migration.ResultsOur results indicated that there was no significant difference of the expression of WNT 1、 WNT 2b, WNT 3a, WNT 4, WNT 9b, WNT 10A and WNT 10B in villi and decidual tissues of normal pregnancy and RSA(P> 0.05), the expression of WNT2 and WNT3 in the recurrent abortion decidual tissue decreased significantly (P < 0.01), however there is no difference in the villus tissues (P> 0.05).What’s more, In co-cultivation experiments of with WNT 2 and WNT 3 with HTR-8/SVneo cell, MMP-2,MMP-9 and CyclinDl epxression significantly increased at both mRNA and protein levels, The trophoblat Cell ability of proliferation and migration increased significantly compared the Control group.ConclusionsOur study suggested that the expression of WNT2 and WNT3 was decresed in the RSA decidual tissue, co-cultivation experiments of WNT 2 and WNT 3 with HTR-8/SVneo cell, confirmed that the Wnt/β-catenin pathway can be activated, over expression the WNT 2 and WNT 3 can proved the proliferation and migration of HTR-8/SVneo cell. Therefore we speculate that the abnormal expression of WNT 2 and WNT3 in the decidual tissue of RSA makes the trophoblast cell’s ability of proliferation and migration decreaseed, which result in the abortion.