以滋养干细胞为模型研究NLRP7调控印迹基因甲基化导致双亲来源完全性葡萄胎发生的分子机制

Hydatidiform moles are abnormally developing pregnancies with trophoblastic hyperproliferation and defective fetal development. Hydatidiform mole occurs recurrently in biparent complete hydatidiform (BiCHM) patients even after they got married with different husband. However, the molecular mechanism of BiCHM remains elusive. It has been widely proved that NLRP7 mutations frequently occur in BiCHM patients. NLRP7 can bind to YY1 in the nuclear and modulate the methylation of imprinted genes. Dysregulation of imprinted genes play a crucial role in hydatidiform mole and many imprinted genes have been proved to be pivotal for trophoblast stem cell differentiation and placenta development. Presumably, NLRP7 mutations, abnormal imprinted genes methylation, and aberrant trophoblast stem cells differentiation may contribute to the occurrence of BiCHM. However, it remains unclear which imprinted genes are required for trophoblast differentiation and how these imprinted genes are regulated by NLRP7. The applicant of this project derived maternal Dnmt3L knockout trophoblast stem cell line. Utilizing this trophoblast stem cell line with defective maternal imprinted genes, this study aim to analyze trophoblast stem cell differentiation-related imprinted genes through restoring individual imprinted genes, further investigate the regulation of NLRP7 on the methylation of these imprinted genes, and elucidate the molecular mechanism of BiCHM.

葡萄胎是一种异常妊娠，表现为滋养细胞过度增生及胚胎发育缺陷。其中，双亲来源的完全性葡萄胎（BiCHM）患者与不同伴侣反复发生葡萄胎，然而其发病机制不清。BiCHM患者常检出NLRP7突变，而NLRP7被发现在细胞核内与YY1因子结合调控某些印迹基因甲基化。印迹基因缺陷是葡萄胎重要发病机制之一，很多印迹基因已被证实与滋养干细胞分化及胎盘发育密切相关。由此推测，NLRP7突变—印迹基因甲基化异常—滋养干细胞分化异常可能是BiCHM发生的关键机制。然而，哪些印迹基因参与滋养干细胞分化，NLRP7突变如何调控这些印迹基因导致BiCHM目前均不清楚。申请人前期构建小鼠母系Dnmt3L敲除滋养干细胞，利用这一细胞印迹基因甲基化及表达缺失的特性，本项目通过逐一恢复印迹基因的表达，以明确滋养干细胞分化相关印迹基因，深入研究NLRP7对这些印迹基因甲基化及表达的调控，从而阐明BiCHM发生的分子机制。

葡萄胎（Hydatidiform Mole，HM）是一种葡萄样良性滋养细胞肿瘤，表现为弥漫性滋养细胞增生和胚胎发育异常。葡萄胎按组织病理学和分子遗传学分二类：部分性葡萄胎（partial hydatidiform mole，PHM）和完全性葡萄胎（complete hydatidiform mole，CHM），其中完全性葡萄胎又分为父系来源完全性葡萄胎（androgenetic CHM, AnCHM）及双亲来源完全性葡萄胎（biparent complete hydatidiform, BiCHM)。葡萄胎常继发其他的妊娠滋养细胞疾病，如绒癌及胎盘部位滋养细胞肿瘤。有研究推测，葡萄胎、侵袭性葡萄胎、中间性滋养细胞肿瘤、绒毛膜癌可能是同一疾病的不同发展阶段，葡萄胎、妊娠滋养细胞肿瘤有着密切关联，滋养干细胞异常分化是根本的发生机制。PSTT病人对化疗敏感性差，一经诊断，需要立即切除子宫，但部分女性即使进行子宫切除，也会出现广泛转移并导致死亡；而临床上出现的难治性绒癌往往迅速进展，造成病人的死亡。因此，对于阐明葡萄胎的发生的分子机制，及其与胎盘部位滋养细胞肿瘤及绒癌发生的机制，对于探索新的治疗手段，改善病人预后具有重要意义。.该项目深入研究发现，NLRP7可能通过影响KHDC3L与YY1结合影响基因甲基化调控，导致葡萄胎的发生；NLRP7下游分子Lin28通过调控Let7a表达调控滋养细胞的增殖，参与滋养细胞的分化；滋养细胞SALL4处于高表达状态，SALL4过度表达促进下游MyC通路的活化，促进滋养细胞的异常增殖，同时SALL4也促进β-catenin在滋养细胞细胞核内的表达，提示SALL4可能通过调控β-catenin及WNT信号通路的活化促进Lin28的活化及滋养细胞的分化；DNMT3L与DNMT3A2结合并抑制其降解，恢复DNMT3A2表达水平，促DNA甲基化及胚胎发育；肿瘤微环境PSTT病灶CAFs通过表达EGR1/Ang-1促进滋养细胞血管形成。上述工作已经发表于Nucleic Acids Res. Frontiers in Oncology, Exp Cell Res., Reprod Sci.，Reprod Biomed Online，ABBS, Cancer Cell International等杂志。