GPER/SphK通路介导雌二醇的子宫内膜癌 “非转录效应”研究

Estradiol(E2) paly biological effects not only by nuclear pathway but also by non-nuclear pathway. But non-nuclear effects of E2 are unclear. Our previous study showed serum E2 levels of endometrial cancer patients(ER negtive expressions) were higher than controls.It was found low ER expression and GPER positive expression in endomtrial cancer HEC-1A cells.E2 induced cell proliferation,ERK activation and inhibited cell apoptosis. These effects were blocked by PGER inhibitor G15 and SphK inhibitor DMS. This project wants to exam the levels of serum E2 and the signal molecules expressions and activity of the tissues of endometrial cancer and controls.In vivo, down-regulations of GPER and SphK1/2 expressions will be performed by transfecting GPER, SphK1 and SphK2 shRNA to down-regulate aim genes.The effects of GPER and SphK1/2 and their downstream signal pathways will by studied after E2 treatment.In vitro, the effects of down-regulation of GPER and SphK1/2 expressions will be studied in nude mice.The volumes and weights of tumors in HEC-1A-shGPER, HEC-1A-shSphK1 and HEC-1A-shSphK2 groups will be observed and the impossible signal moelcules will be examed. If excepted results are obtained, important clues will be offered to clarify molecule mechanism of formation and development of endometrial cancer. New methods and targets will be supplied to cure endometrial cancer.

雌二醇（E2）不仅通过细胞核受体（ER）发挥基因组效应，也能通过其他途径发挥“非基因效应”，但E2的非基因效应作用途径尚不明确。前期研究显示ER阴性内膜癌患者血浆E2高于对照组，E2能诱导ER低表达PGER高表达的内膜癌HEC-1A细胞增殖、ERK活化及抑制凋亡，PGER抑制剂及SphK抑制剂能抑制上述效应。本课题拟进行内膜癌患者血浆E2及组织中相关分子表达及活性测定并进行相关性研究。体外实验应用慢病毒敲除GPER及SphK1/2，建立稳定敲除细胞株。E2作用后检测GPER/SphK1和（或）SphK2在其中发挥的作用,研究可能的下游信号通路。体内实验建立裸鼠成瘤模型，E2刺激后检测移植瘤进展并进行相关分子检测。本课题若能获得预期结果，证实E2/GPER/SphK非基因效应促进子宫内膜癌生物学进程，对阐明子宫内膜癌发病机理有重要意义，为恶性程度更高的ER阴性子宫内膜癌防治提供新的靶点和依据

子宫内膜癌作为常见的妇科肿瘤之一，大部分病例其发生发展与患者雌二醇（E2，estradiol）水平密切相关。E2既可以通过激活位于细胞核的雌激素受体（estrogen receptor，ER）发挥转录效应，也可通过激活位于细胞膜的G蛋白偶联雌激素受体（G protein coupled estrogen receptor，GPER）发挥非转录效应。但E2的非转录效应作用途径尚不明确。.本研究首先对子宫内膜癌患者血浆E2水平以及肿瘤组织GPER、ER表达、SphK活性进行了检测，以确定子宫内膜癌发生与GPER、SphK活性的相关性。同时采用HEC-1A子宫内膜癌细胞培养的方法，观察了E2对HEC-1A增殖、侵袭、迁移的影响以及E2对细胞SphK活性、SphK表达以及ERK磷酸化、CyclinD1、CyclinE1、MMP-9表达的影响。此外，应用慢病毒敲除GPER及SphK1/2，建立稳定敲除细胞株，观察敲减GPER、SphK1/2对E2促增殖、迁移、侵袭作用的影响。体内部分研究，采用HEC-1A皮下接种的方法建立裸鼠移植性肿瘤模型，观察E2对整体动物移植瘤的影响并进一步探索其分子机制。.临床研究结果显示，子宫内膜癌组织GPER阳性表达与肿瘤FIGO分期、组织分化、肌层浸润密切相关。GPER阳性患者SphK活性明显高于阴性表达照组。HEC-1A子宫内膜癌细胞株体内体外研究证实，E2可促进子宫内膜癌的增殖迁移及侵袭，并增加SphK活性，增加ERK1/2磷酸化，并增加CyclinD1、CyclinE1、MMP-9表达。而GPER阻断剂G-15、SphK抑制剂DMS、ERK抑制剂PD98059可抑制E2的上述作用。敲减GPER、SphK1亦可抑制E2的促增殖、迁移及侵袭作用。裸鼠实验显示，E2可促进移植性肿瘤生长，增加其瘤重及肿瘤体积。并促进肿瘤组织ERK1/2磷酸化及CyclinD1、CyclinE1、MMP-9表达。阻断GPER、抑制SphK或ERK可抑制E2的促肿瘤作用，敲减GPER或SphK1亦可发挥相同的作用，但敲减SphK2对肿瘤增殖无影响。.本研究进一步明确了E2促进GPER阳性子宫内膜癌增殖的机制，E2可通过GPER/SphK1/ERK途径促进子宫内膜癌生长，而GPER、SphK1可以作为治疗子宫内膜癌的新靶点。