溶酶体L型电压门控钙通道Cav1.3调节自噬参与子宫内膜癌非基因转录效应的机制研究

The pathogenesis endometrial carcinoma is closely related to estrogen. The incidence of endometrial cancer is closely related with genomic effect and nongenomic effect of estrogen. Our previous study found that calcium is an important "second messenger", inducing non-genomic effects of endometrial carcinoma, L-type voltage-gated calcium channel Cav1.3 is a key regulatory protein, which promotes the proliferation and metastasis of endometrial carcinoma，and the Cav1.3 participate in the regulation of autophagy. The lysosome is the terminal of autophagy. However, the role of lysosomal calcium channel Cav1.3 in non-genomic effect of estrogen-induced calcium release in endometrial carcinoma has not been reported. This project intends to do research with molecular biology as follows: Firstly, Laser confocal techniques were used to study the regulation of lysosomal calcium channel Cav1.3 stimulated with estrogen in endometrial cancer cells; Secondly, to research the effects of lysosomal calcium channel Cav1.3 on the stability and function of lysosome in endometrial carcinoma cells by knocking out or overexpressing Cav1.3 with CRISPR/Cas9; Thirdly, to detect the effect of calcium channel Cav1.3 on autophagy and related signaling pathways，and to verify the role of lysosomal calcium channel in the development of endometrial carcinoma in animal models. This study will elucidate that estrogen may participate in non-genomic effects of endometrial cancer by regulating calcium channels localized in lysosomes, and provide information for finding new targets for organelle ion targeted therapy.

子宫内膜癌发病与雌激素密切相关，雌激素对子宫内膜癌作用存在基因转录效应和非基因转录效应。本课题组前期研究发现钙离子是雌激素非基因转录中重要的“第二信使”，L型电压门控钙通道Cav1.3是关键的调控蛋白，促进子宫内膜癌增殖、转移，参与调节细胞自噬，而溶酶体是细胞自噬的终点，溶酶体膜钙通道Cav1.3对雌激素引起的钙释放及其调节机制尚不明确。本课题拟：①应用激光共聚焦等技术研究雌激素对子宫内膜癌细胞溶酶体钙通道Cav1.3的调控；②利用CRISPR/Cas9敲除或过表达Cav1.3，研究Cav1.3对溶酶体稳定性及与自噬体融合的影响；③研究Cav1.3对子宫内膜癌细胞自噬的调节及相关信号通路，动物实验验证溶酶体钙通道对子宫内膜癌发生发展的作用。以阐明雌激素可能通过调控溶酶体钙通道Cav1.3调节自噬，参与非基因转录效应、促进子宫内膜癌细胞增殖转移，为寻找细胞器离子靶向治疗新靶点提供理论依据。

项目基本遵循原计划执行并完成了预期的研究目标，同时将基础实验与临床数据结合，研究了雌激素导致胞内Ca2+水平变化的来源，认为血清钙离子与子宫内膜癌的临床病理特征存在相关性，雌激素的非基因转录效应与胞外Ca2+水平及关键钙离子通道Cav1.3密切相关，并能通过影响细胞器钙稳态，最终调控细胞生理功能，介导内膜癌的发生与发展；通过进一步完善雌激素在子宫内膜癌中非基因转录效应相关作用机制，找到非基因转录效应与基因转录效应的联络点，为子宫内膜癌的预防、诊断及靶向治疗提供新的思路。