核转录因子NRF3通过调控氧化应激促进膀胱癌发生发展的机制研究

Recently, with the development of molecular biology and proteomics, the specific protein factors related to the development and progression of bladder cancer become a research hotspot; especially the discovery of marker genes could improve the sensitivity and specificity of early diagnosis and treatment of bladder cancer, and could be a target for finding new anticancer drugs and methods. Our previous studies have found that overexpression of CCDC34 in bladder cancer could activate the expression of Nuclear Factor-Erythroid 2-related factor 3 (NRF3) through MAPK and AKT signaling pathways, promoting cell carcinogenesis. Besides, NRF3 was involved in the regulation of antioxidant genes and phase II detoxification genes. So NRF3 is of great significance in the biological development and clinical manifestations of bladder cancer, and there has been no relevant study yet. Based on our already established bladder cancer specimen library and clinicopathological database with follow-ups, this project aims to fully elucidate the biological functions of NRF3 in bladder cancer cells proliferation, migration and invasion; and its clinical significance from the three levels of cells, tissues and animals. Besides, we will further screen and identify important target genes and signaling pathways that NRF3 regulates; and direct interaction proteins of NRF3, thus elucidating the mechanisms of NRF3 promoting the progression of bladder cancer through regulating oxidative stress. Our research will help to improve the pathogenesis of bladder cancer and provide important theoretical basis and new targets for the diagnosis and treatment of bladder cancer.

随着分子生物学和蛋白组学技术的不断发展及研究的深入，与膀胱癌发生发展相关的特异性蛋白因子成为研究热点，标志基因的发现可提高膀胱癌早期诊断及治疗的敏感性和特异性，可成为寻找新抗肿瘤药物和方法的靶标。前期研究发现，膀胱癌高表达基因CCDC34可通过MAPK和AKT信号通路激活核因子E2相关因子3（NRF3）的表达使细胞发生癌变；NRF3参与了对抗氧化和Ⅱ相解毒基因的调节，且在膀胱癌生物学发生发展及其临床表象中具有重要意义，目前国内外尚未见相关报道。基于本中心已建立的膀胱癌标本库和临床病理及随访数据库的基础上，本研究拟：①从组织、细胞和动物水平阐明NRF3促进膀胱癌细胞增殖和侵袭迁移的生物学功能及其临床价值；②筛选鉴定NRF3调控的重要靶基因、信号通路及其互作蛋白分子，明确NRF3调控氧化应激促进膀胱癌进展的机制。本课题将有助于完善膀胱癌的发病机制，为膀胱癌的诊断和治疗提供重要理论依据和新靶点。

NFE2L3 (NRF3)作为一种受严格调控和转录后修饰的转录因子，参与了多种细胞的生物学行为过程，如肿瘤的发生、细胞应激反应、分化和炎症反应等；目前在肿瘤研究中，其同源基因NFE2L2 (NRF2)已被广泛研究，但NRF3的功能和调控机制还没有得到深入的阐明，尤其是在泌尿系肿瘤的发生发展和治疗中。在本项目研究中，我们结合TCGA数据库和本中心膀胱癌临床病理及生存随访数据库，发现NRF3在膀胱癌组织和细胞中的表达显著高于其配对癌旁组织和正常膀胱上皮细胞，NRF3高表达患者的肿瘤分期更高，更易发生远处转移，且NRF3高表达是患者OS和MFS的独立预后因素。通过体内外功能实验表明，NRF3敲低后可以显著抑制膀胱癌细胞的增殖、迁移和侵袭，并诱导肿瘤细胞凋亡增加和细胞周期阻滞于G1期；功能回复实验表明NRF3过表达在体内外均可以恢复促进膀胱癌细胞迁移和侵袭的表型。通过对NRF3敲低和过表达的膀胱癌细胞分别进行转录组测序联合定量蛋白组学检测分析，我们发现NRF3调控影响的差异基因主要富集于肿瘤进展和转移相关的基因/信号通路，如细胞黏附通路、肌动蛋白细胞骨架调节通路、ECM受体通路等；NRF3过表达促进了膀胱癌细胞的上皮-间质转化，紧密连接蛋白ZO-1和上皮标记E-cadherin的表达减少，转录因子Snail1和Snail2表达升高；进一步分别阐明了受NRF3调控的下游分子TAGLN、MMP14和P4HB在促进膀胱癌转移中的功能和分子作用机制。通过蛋白组联合COIP-MS筛选获得了与NRF3互作的蛋白分子NDRG1和FBXW7。此外，我们发现NRF3在膀胱癌顺铂和吉西他滨耐药细胞系中均显著高表达，我们后续将继续探究明确NRF3在膀胱癌化疗抵抗中的分子作用机制。综上，NRF3促进了膀胱癌的进展转移，可作为膀胱癌诊断和转移的有价值的肿瘤标志物，并且是膀胱癌化疗抵抗的一个重要潜在靶点。