Wnt/β-catenin信号通路甲基化修饰调控滋养细胞侵袭在子痫前期发病中的机制研究

Placenta shallow implantation induced by decreased invasion of trophoblasts is an important pathological character of preeclampsia(PE). The activation of Wnt/β-catenin signaling pathway can promote the invasion of trophoblasts. Our previous results showed that the expression of some ligands of Wnt were decreased in placentas of PE patients, but the expression of Wnt inhibitors, DKK1,SFRP4,WIF-1, were increased. The methylation level of SFRP4 was lower in PE group than in the control group. We hypothesize that the decreased activation of Wnt/β-catenin signaling pathway is induced by the methylation modification, which suppresses the invasion of trophoblasts and facilitates the occurrence and development of PE. To test our hypothesis, we propose to measure the methylation in the promoter regions of 14 associated factors and 8 inhibitors of Wnt in placentas of PE and normal control by using bisulfite sequencing combined with high-throughput sequencing platform. To verify the correlation of the methylation modification with the invasive ability of trophoblasts at cellullar level, methylation levels will be compared among different cell lines including villi trophoblasts with no invasive ability, primary extravilli trophoblasts with invasive ability and chorionic carcinoma cell lines. The established animal models will be used to examine whether the decreased activation of Wnt/β-catenin signaling pathway can lead to the associated symptoms of PE. Our study aims are to provide new theoretical basis and molecular markers for the pathogenesis of preeclampsia.

滋养细胞侵袭力下降引发胎盘浅着床是子痫前期重要的病理特征，且Wnt/β-catenin信号通路激活可促进滋养细胞侵袭。我们前期研究发现: 某些Wnt配体在子痫前期胎盘组织中表达下调，而Wnt抑制因子DKK1、SFRP4、WIF-1表达上调，且SFRP4的甲基化水平低于正常对照组。因此假设:Wnt/β-catenin信号通路通过甲基化修饰下调通路活性，抑制滋养细胞的侵袭，参与子痫前期的发生、发展。为验证此假设：拟采用亚硫酸盐测序结合高通量测序平台在子痫前期胎盘组织中检测14种Wnt相关因子及8种Wnt抑制因子基因启动子甲基化状态；利用原代培养的无侵袭性的绒毛滋养细胞和具有侵袭性的绒毛外滋养细胞及绒毛膜癌细胞系，在细胞水平验证Wnt通路甲基化修饰与滋养细胞侵袭的相关性; 利用已建立的子痫前期动物模型验证Wnt通路活性下降是否会引发孕鼠子痫前期相关症状。为子痫前期的发病机制提供新理论依据。

滋养细胞侵袭力下降引发胎盘浅着床是子痫前期（preeclampsia，PE）重要的病理特征，本项目前期研究提示WNT/β-catenin信号通路和PE密切相关，为明确WNT相关因子的甲基化状态对胎盘滋养细胞侵袭力的影响及其与PE的相关性，本项目采用包含85万探针的高通量甲基化芯片在PE、特异性早产和正常对照胎盘组织中检测14种WNT配体及8种WNT抑制因子基因启动子区域甲基化状态。结果发现（1）多数WNT因子的甲基化状态与孕周相关。与正产孕妇胎盘相比，PE组WNT相关因子多处于高甲基化状态，共87个差异性位点（78个位点高甲基化，9个位点低甲基化），而与同孕周的特异性早产胎盘组织相比，仅有25个差异性位点（14个位点高甲基化，11个位点低甲基化）。（2）所有差异性位点中仅有WNT3基因的低甲基化状态在PE组和早产组中有显著差异，在PE与早产组之间和早产与正产组之间无差异，提示与PE密切相关；通过进一步扩大临床样本量分析，发现WNT配体在PE胎盘中多表达下调（WNT1、WNT2、WNT2b、β-catenin），WNT抑制因子在PE胎盘中多表达上调（DKK1和WIF1），WNT信号通路在PE胎盘中整体处于抑制状态，而WNT3基因却在PE胎盘中表达上调，与WNT3基因在PE中的低甲基化相一致，提示胎盘中WNT3基因启动子区域的甲基化改变影响WNT3基因的表达。细胞实验中证实WNT3基因表达量增加确实可以促进滋养细胞侵袭，但在L-NAME诱导的妊娠期高血压小鼠模型中，WNT3基因的表达量无变化，提示PE胎盘组织中滋养细胞侵袭力减弱机制中WNT3配体可能作为一种负调控作用存在。本研究为PE的发病机制提供了新的理论依据。