转录因子GRHL3调控G蛋白偶联受体108在皮肤鳞癌发生中的作用及其机制

GRHL3, as a transcription factor, positively and negatively regulates some different target genes for controlling growth, differentiation, migration and invasion of epidermis cells during embryonic development. Recently, GRHL3 becomes a popular molecule involving in tumorigenesis and progression for skin squamous cell carcinoma. We have proved that negative regulation of GRHL3 could inhibit expression of the target genes and control migration and invasion of cancer cells; also it was reported that positive regulation of GRHL3 is related to apoptosis and proliferation of cancer cells. The G protein coupled-receptor 108 (GPR108) is a member of the GPR super family and locates at the membrane of Golgi’s apparatus. We previously found that expression of GPR108 was consistent with GRHL3 expression profile in skin cancer tissues. Importantly, GRHL3 could increase activity of the GPR108 gene promoter. So GPR108 may be a target gene regulated positively by GRHL3, even it is less known about it's functions. Additionally, we found GPR108 knockout cells are resistant to apoptosis induced by ER stress. We guess GRHL3 down-expression in skin cancer cells may lead to down-regulation of GPR108, and loss membrane functions of Golgi’s apparatus, then inhibit ATF6 activation. So cancer cells may be resistant to sub-lethal ER stress /UPR and be apt to proliferation because of losing apoptosis. In this project, we would use skin cancer tissues, GPR108 knockout mice, molecular and cellular technologies to explore positive regulation mechanism of GPR108 expression regulated by GRHL3, to find functions and mechanisms of GPR108 while skin squamous cell carcinoma occurs. The project may be useful to find a new potential target site for preventive treatment of skin squamous cell carcinoma.

在胚胎发育中转录因子GRHL3调控不同靶基因参与表皮细胞增殖、分化和迁移，其在皮肤鳞癌中作用备受关注。我们已证实其负调控抑制靶基因表达与鳞癌细胞迁移和侵袭有关；有报道其正调控的靶基因与细胞凋亡和增殖有关。我们发现鳞癌早期GRHL3减少伴有定位于高尔基器膜的G蛋白偶联受体GPR108表达降低，并已证实GRHL3能增强GPR108启动子活性。故GPR108可能是GRHL3的正调控靶基因，但功能未知。前期还发现内质网应激时GPR108缺失细胞耐受细胞凋亡。推测皮肤鳞癌发生因GRHL3表达下调导致GPR108减少，高尔基器膜功能障碍抑制ATF6活化，细胞耐受亚致死性内质网应激，抑制凋亡而促进细胞增殖。本项目拟利用皮肤鳞癌组织和GPR108基因敲除小鼠模型，运用分子和细胞生物技术，研究GRHL3正调控GPR108表达、GPR108的功能及其在皮肤鳞癌发生的作用和机制，为防治皮肤鳞癌寻找新型药物靶点。

背景：作为一种7次跨膜蛋白，GPR108的功能及其在肿瘤中的作用和机制不清楚。转录因子GRHL3调控不同靶基因参与表皮细胞及癌细胞的增殖、分化和迁移。结构分析发现gpr108基因启动子含潜在GRHL3结合位点。本项目旨在研究GRHL3调控gpr108基因表达的机制、探讨GPR108的功能及其在相关疾病中的作用和机制。.方法：利用生物信息学分析GRHL3和GPR108的表达规律并在细胞和组织水平利用IHC、WB验证；利用分子克隆技术研究GRHL3调控GPR108表达的机制；分子细胞生物学方法探讨GPR108细胞内分布及其靶向输送的机制；利用Gpr108-/-的免疫细胞和非免疫细胞等研究GPR108的功能；利用Gpr108-/-小鼠皮肤肿瘤模型、临床病例组织标本或细胞系等，采用IHC、WB等方法，探讨GPR108及其下游分子在皮肤肿瘤和炎症性疾病等在发生发展中的作用和机制。.内容和结果：发现多种肿瘤细胞系中GRHL3与GPR108表达存在负相关的表达趋势；证实GRHL3特异结合gpr108基因启动子区的保守序列并抑制gpr108表达；证实GPR108在细胞内分布和靶向输送依赖于SNX27，提示SNX27可作为GPR相关肿瘤的治疗靶点；发现2种GPR108特异性结合的小分子EE和EA，能调控互作分子MyD88表达；首次报道GPR108功能是参与Toll样受体介导的炎症反应，其机制是调节NF-kB信号通路，并影响IL-6、IL-1β、TNFɑ和IFNɣ等炎症因子分泌。.皮肤肿瘤模型研究证实GPR108的缺失有利于抑制肿瘤的发生，其机制是与活化NF-kB/TNFɑ信号通路、促进细胞凋亡有关；炎症性疾病研究中发现GPR108调控的下游炎症因子IL-6、IL-1β可活化STAT3和NF-kB，调节p53、p21表达，进而促进血管平滑肌细胞发生衰老相关的钙磷沉积及慢性肾病的血管钙化；IL-6活化MEK/ERK、JAK/STAT3、PI3K/AKT等信号通路，促进转录因子LMO4表达，调控KC细胞的增殖和分化且参与银屑病的发生发展；IL-1β可诱发肾小管上皮细胞发生EMT，参与慢性肾病并发肾脏纤维化，该作用受NF-kB/TIR8的负反馈调节。这些结果为进一步研究GPR108的功能奠定了基础。.该项目支持的成果有发表的核心期刊论文2篇、SCI论文7篇；培养的硕士生9名、博士生1名。