SREBP1介导Hippo通路在子宫内膜样腺癌孕激素抵抗机制的研究

The incidence rate of endometrial adenocarcinoma (EAC) is constantly growing, and the age of onset is the trend of younger. High-dose progesterone is an effective endocrine treatment for young EAC patients with the demand of preserving fertility. However, up to 30 percent of patients are not sensitive to progesterone or develop resistance to progesterone during the treatment. And the concrete mechanisms are not clear. Some studies have shown that sterol regulatory element binding protein 1(SREBP1), which is highly expressed in many human malignancies, acts as an oncogene by regulating lipids metabolism. We found that the overexpression of SREBP1 enhances the occurrence and development of EAC, while SREBP1, YAP/TAZ and MAGL all strongly express in progesterone-resistant Ishikawa cells which have been successfully established during preliminary experiment. Inversely, inhibition of SREBP1 renders progesterone-resistant Ishikawa cells more susceptible to progesterone treatment, following the down regulation of YAP/TAZ and MAGL. We hypothesized that the overexpression of SREBP1 promotes the proliferation and migration of progesterone-resistant Ishikawa cells by upregulating YAP/TAZ and MAGL expression. In this study, knocking down or overexpressing SREBP1 in EAC cells, luciferase assay and nude mice xenograft model etc. will be performed to further explore the mechanisms of MAGL regulated by SREBP1 through hippo pathway in progesterone resistance development. It may provide potential therapeutic targets for the treatment of young EAC patients with progesterone resistance.

子宫内膜样腺癌（Endometrial adenocarcinoma，EAC）发病率逐年增加，日益年轻化，孕激素是年轻EAC患者保留生育功能的主要治疗手段，然逾30%患者对孕激素不敏感或在治疗过程中产生孕激素抵抗，具体机制不清。研究表明SREBP1可通过调节脂代谢在恶性肿瘤中发挥促癌作用。前期证实SREBP1可促进EAC的发生发展，我们在建立的孕激素抵抗细胞株（Ish/MPA）中发现SREBP1，YAP/TAZ，MAGL一致高表达，敲除SREBP1可改善Ish/MPA细胞株对孕激素的敏感性，下调YAP/TAZ及MAGL，初步表明SREBP1通过YAP/TAZ及MAGL增强细胞增殖迁移能力，促进细胞在孕激素治疗下的生存，诱发孕激素抵抗。本课题拟通过基因干扰、双荧光素酶报告、裸鼠移植瘤等体内外实验研究SREBP1介导Hippo通路调控MAGL与孕激素抵抗的相关机制，以期解决临床孕激素抵抗问题。

课题按照预定计划进行，首先采用免疫组织化学技术，检测孕激素治疗前后子宫内膜组织中SREBP1、YAP/TAZ、MAGL、PR的蛋白表达水平及分布情况，明确SREBP1与子宫内膜癌孕激素抵抗的发生具有临床上的相关性；选择孕激素敏感的Ishikawa细胞、等浓度孕激素处理长期诱导形成的孕激素抵抗 Ishikawa 细胞（Ishikawa-MR）及原发孕激素抵抗的HEC-1A细胞,利用shRNA 干扰和（或）慢病毒过表达SREBP1，构建稳定细胞系。采用MTT实验、Edu增殖实验、流式凋亡实验、Western Blot技术及体内成瘤实验检测浓度梯度孕激素作用下SREBP1表达对子宫内膜样腺癌细胞生物学行为的影响，建立子宫内膜样腺癌孕激素抵抗裸鼠移植瘤模型，证明SREBP1 抑制剂作为孕激素抵抗辅助治疗的可行性；除此之外，利用Western Blot技术检测Ishikawa和Ishikawa-MR细胞中YAP、TAZ、MAGL的表达，利用表达载体和 RNA 干扰技术建立YAP/TAZ过表达和低表达（shRNA）稳定细胞系,检测细胞MAGL的表达，揭示Hippo通路和MAGL在孕激素抵抗过程中的相关性。本研究通过验证SREBP1-NFKB通路及Hippo-MAGL通路在子宫内膜癌孕激素抵抗的发生发展中的作用，为进一步研发逆转孕激素抵抗的药物提供理论依据，解决临床孕激素抵抗难题。