GPER1与AMF正向反馈调控：Ⅱ型子宫内膜癌发病假想及其机制

Recent studies have revealed that type Ⅱ endometrial cancer which is lack of estrogen nuclear receptor (ER) expression may also associate with estrogen stimulation, the etiology of type Ⅱ endometrial cancer may, therefore, not be completely estrogen independent, as previously believed. Our previous studies showed that the rapid non-genomic effects induced by conjugation of estrogen and G protein-coupled estrogen receptor 1 (GPER1) promoted type Ⅱ endometrial cancer cell proliferation and invasion. Autocrine motility factor (AMF) secreted by type Ⅱ endometrial cancer cells mediated tumor cells migration and epithelial-mesenchymal transition (EMT) through the MAPK signaling pathway. We presume and obtain preliminary validation of the preliminary experimental results: under the condition of estrogen stimulation, the positive feedback regulation loop between GPER1 and AMF probably accelerate the proliferation of type Ⅱ endometrial cancer cells. On the basis of the preliminary experiments, the present study aims to perform adenovirus-mediated somatic genome editing of AMF by CRISPR/Cas9 in female nude mouse transplanted tumor, and explore the expression of GPER1 after AMF knockout; to analysis GPER1 expression trend under AMF rescue condition; to compare the expression of AMF and GPER1 under estrogen stimulation in vivo. The research will deepen the understanding of the underlying mechanism of poorer prognosis of type Ⅱ endometrial cancer, and provide a new approach of molecular targeted therapy for estrogen.

近期研究提示，雌激素核受体（ER）缺失的Ⅱ型子宫内膜癌（内膜癌）也具有雌激素依赖性，经典的Ⅱ型内膜癌非雌激素致癌理论遭到质疑。课题组前期研究分别证明了：雌激素与G蛋白偶联雌激素受体1（GPER1）结合产生的非基因组快速效应是调控Ⅱ型内膜癌细胞增殖和侵袭的重要途径；Ⅱ型内膜癌细胞产生的自分泌移动因子（AMF）能激活下游MAPK信号通路，诱导肿瘤细胞的EMT。我们推测并得到预实验结果的初步验证：在雌激素影响下，Ⅱ型内膜癌细胞存在的GPER1-AMF相互间正向反馈调控能加速肿瘤增殖。课题将构建移植瘤中AMF被敲除（CRISPR／Cas9技术）的雌性裸鼠模型，观察AMF被敲除后GPER1表达；分析AMF拯救条件下GPER1的表达反弹趋势；比较在雌激素刺激下体内AMF和GPER1表达。研究将会加深我们对Ⅱ型内膜癌预后更为凶险的内在机制理解，为寻找能阻断雌激素作用靶点关键因子的分子靶向治疗提供新思路。

近期研究提示，雌激素核受体（ER）缺失的Ⅱ型子宫内膜癌（内膜癌）也具有雌激素依赖性，经典的Ⅱ型内膜癌非雌激素致癌理论遭到质疑。课题组前期研究分别证明了：雌激素与G蛋白偶联雌激素受体1（GPER1）结合产生的非基因组快速效应是调控Ⅱ型内膜癌细胞增殖和侵袭的重要途径；Ⅱ型内膜癌细胞产生的自分泌移动因子（AMF）能激活下游MAPK信号通路，诱导肿瘤细胞的EMT。前期预实验证实，在雌激素影响下，Ⅱ型内膜癌细胞存在的GPER1-AMF相互间正向反馈调控能加速肿瘤增殖。本研究在预实验的基础上发现：1.与正常子宫内膜相比，AMF和GPR30在内膜癌组织中呈高表达，且两者表达水平呈正相关，提示AMF可能通过E2-GPR30途径促进内膜癌的发展。2.在雌激素及GPR30特异性激动剂G1刺激下，ERα表达阳性及阴性的内膜癌细胞培养上清中AMF分泌均增加，提示AMF的分泌受雌激素调控，且存在除ERα外的其他调控途径。3.利用shRNA干扰内膜癌细胞GPR30的表达能消减雌激素对AMF的分泌诱导作用，提示GPR30介导了雌激素对AMF的表达和分泌调控。4.干扰AMF表达后，通过G蛋白信号通路芯片筛查，发现MAPK-ERK信号通路改变最显著，此外抑制MAPK-ERK信号通路能消减AMF对GPR30的上调作用，提示AMF可激活MAPK信号通路正向反馈调控GPR30的表达。本研究证明了，雌激素通过其膜受体GPER1促进AMF分泌，AMF通过MAPK-ERK信号通路介导可上调GPER1表达，通过这种自分泌正向反馈调节机制影响内膜癌的发生发展。研究成果加深了我们对Ⅱ型内膜癌预后更为凶险的内在机制理解，为寻找能阻断雌激素作用靶点关键因子的分子靶向治疗提供了新思路。