IGF2BP3通过抑制Fbxw7介导的MYC泛素化降解调控膀胱肿瘤增殖的作用及机制研究

Little is known about the regulatory function of oncogene IGF2BP3 on the progressive development of bladder cancer as well as its underlying molecular mechanisms. It is acknowledged that, as a RNA-binding protein, IGF2BP3 increases the expression of its target genes via binding to their mRNAs. Our preliminary experiments indicated that bladder cancer patients with a higher expression of IGF2BP3 had poorer prognosis. And IGF2BP3 was able to regulate tumor proliferation by stabilizing MYC protein. So we raise our hypothesis that IGF2BP3 regulates proliferation of bladder cancer via inhibiting Fbxw7-mediated ubiquitination and degradation of MYC, which will be tested and verified in this project. We have analyzed the correlation between the expression of IGF2BP3 and the prognosis of bladder cancer patients in a retrospective study. Next the regulatory function of IGF2BP3-MYC on tumor proliferation will be studied in vitro and in vivo by using methods like gene overexpression/silence, immunoblot, cell proliferation assay and so on. The underlying regulatory mechanism that IGF2BP3 stabilized MYC protein will be studied by using methods such as construction of molecular clones, transfection and co-immunoprecipitation. The project will for the first time reveal the function of IGF2BP3 on proliferation of bladder cancer as well as its underlying molecular mechanisms, which will provide new insights on its applications of biomarker and therapeutic target. Also, this study will enrich our understanding of the IGF2BP3 that functions independent of its RNA-binding activity.

癌胚基因IGF2BP3，作为RNA结合蛋白，通过结合并稳定mRNA来提高目的基因的表达，然而其对膀胱癌恶性进展的调控作用和分子机制还知之甚少。我们预实验提示IGF2BP3高表达的膀胱癌患者预后较差，且可能通过稳定MYC蛋白来调控细胞增殖。我们提出假说“IGF2BP3通过抑制Fbxw7介导的MYC泛素化降解来调控膀胱肿瘤细胞的增殖”。为验证该假说，我们已完成预后相关性的回顾性研究；接下来拟在体外细胞系培养和体内大鼠原位肿瘤模型上，通过基因沉默/过表达、免疫印迹、免疫组化、细胞增殖检测等技术研究IGF2BP3-MYC轴对肿瘤细胞增殖的调控作用；通过分子克隆、细胞转染、免疫共沉淀等技术研究IGF2BP3对MYC蛋白稳定性的调控机制。本课题首次揭示IGF2BP3对膀胱肿瘤细胞增殖的调控作用和分子机制，为膀胱癌的标志物和治疗靶点选择提供新思路；丰富了对其作为RNA结合蛋白以外其他功能的认识。

IGF2BP3作为癌基因，其对膀胱癌细胞恶性进展的调控作用和分子机制还知之甚少。传统观点认为，IGF2BP3作为RNA结合蛋白，通过结合并稳定mRNA来提高目的基因的表达水平。我们对TCGA库中408例MIBC患者的回顾性研究发现IGF2BP3高表达的膀胱癌肿瘤分期分级更高、患者预后更差。对本院20例 MIBC样本的免疫组化检测和蛋白组学分析发现IGF2BP3的表达程度和肿瘤增殖活跃程度正相关。在尿路上皮癌细胞系RT112和UMUC-3上，通过IGF2BP3基因沉默/过表达、免疫印迹、细胞增殖检测等方法发现IGF2BP3通过正向调节MYC表达来调控肿瘤的增殖，表现加快的细胞周期进展和更强的克隆形成能力。在体内小鼠皮下移植瘤肿瘤模型上，发现IGF2BP3高表达的膀胱肿瘤生长速度更快，肿瘤细胞MYC的表达和Ki67阳性的比例更高。从机制上，IGF2BP3不仅提高MYC的mRNA水平，并且通过抑制泛素化降解途径增加了MYC蛋白的稳定性。Genemania和HitPredict软件预测IGF2BP3和MYC间存在物理结合，免疫共沉淀方法证实了IGF2BP3通过和Fbxw7竞争性结合到MYC的TAD区域，从而阻止Fbxw7介导的MYC泛素化降解。本课题首次揭示IGF2BP3通过抑制Fbxw7介导的MYC泛素化降解促进膀胱癌的增殖，为膀胱癌的预后标志物和治疗靶点选择提供新思路，丰富对IGF2BP3作为RNA结合蛋白以外的其他功能的认识。