COPB2及其互作蛋白在肌层浸润性膀胱癌进展中的机制研究

Muscle invasive bladder cancer (MIBC) presents high rate of recurrence and progression in urinary system. There has been a lack of molecular markers for clinical diagnosis and prognosis predictors of MIBC up to now. Previously, we used proteomics tools to identify differential expressed protein in MIBC. The most highly expressed protein, coatomer subunit beta 2 (COPB2), has been indicated to play a central role in bladder cancer cell proliferation, invasion and apoptosis. Additionally, we utilized gene chip to explore the interacting protein of COPB2. Combined with immunoprecipitation, we demonstrated that nuclear protein 1 (NUPR1) was the mutual protein of COPB2. Our functional experiments suggested that NUPR1 was also involved in the proliferation of bladder cancer. Based on these findings, we presumed that both COPB2 and NUPR1 participate in the progress of MIBC. To verify this hypothesis, we are going to: (1) explore the expression of COPB2/NUPR1 in MIBC, non-MIBC and control tissues by immunohistochemistry; (2) confirm whether both of them participate in MIBC proliferation, invasion and metastasis via in vitro experiments; (3) conduct animal experiments to verify the results of in vitro ones; (4) use Mass spectrometry combined with functional complementation to determine the specific mechanisms in the process of MIBC. The aim of the present study was to identify an independent molecular marker to predict bladder cancer outcome. It is also expected to provide a theoretical basis for new targeted drugs to treat MIBC.

肌层浸润性膀胱癌（MIBC）是膀胱癌中恶性度较高的肿瘤，目前仍缺乏准确诊断MIBC的分子标记物及评价预后的预测因子。课题组前期通过对MIBC差异蛋白分析发现泡膜蛋白COPB2异常高表达，且与膀胱癌增殖、凋亡、侵袭密切相关；通过基因芯片预测协同互作小分子蛋白NUPR1，免疫共沉淀确定两者互相结合，功能学实验提示NUPR1亦参与膀胱癌的增殖。我们推测COPB2与NUPR1互相作用共同参与MIBC的进展，拟进一步1、收集临床MIBC组织及对照，免疫组化检测COPB2与NUPR1表达差异，分析两者表达与膀胱癌患者临床特征及术后复发、存活率和化疗药物敏感性的相关性；2、完善细胞功能学研究，明确两者参与促进膀胱癌进展、侵袭和转移的机制；3、动物实验验证细胞学功能；4、通过芯片、质谱及大数据手段分析COPB2与NUPR1促进膀胱癌进展及转移的关键信号机制。通过上述研究为MIBC诊断、预后评价提供新策略。

膀胱癌，特别是肌层浸润性膀胱癌是目前临床研究热点。本课题基于蛋白组学以及表达谱芯片分析鉴定的NUPR1蛋白，对其进行临床、功能和机制研究。.1、NUPR1在膀胱癌增殖、迁移与侵袭中的作用和机制。.通过临床-细胞，动物-分子机制研究证实：①通过对80例膀胱癌患者的肿瘤和癌旁组织进行免疫组化染色研究，证实了NUPR1在膀胱癌组织中高表达；②通过对膀胱癌细胞株（T24/5637）研究，利用细胞凋亡、Transwell迁移实验及Transwell侵袭实验，证实NUPR1能促进膀胱癌增殖、迁移与侵袭；③通过裸鼠皮下成瘤实验，注射T24细胞，证实NUPR1能促进瘤体的生长；④通过表达谱芯片分析，证实NUPR1最显著的下游分子是脂多糖；⑤通过GSEA及Western blot，证实NUPR1促进膀胱癌增殖是通过影响上皮-间质转化、细胞凋亡、细胞周期、炎症和IL6-JAK-STAT3等信号通路实现的；⑥此部分研究内容已发表SCI文章1篇。.2、NUPR1表达与膀胱癌临床特征指标的相关性。.①NUPR1在膀胱癌组织中高表达；②NUPR1高表达与肿瘤的临床分期、术后复发呈正相关。③ 通过大数据分析结合功能实验证实：NUPR1与上皮-间充质转化相关。.3、膀胱癌中NUPR1的作用以及与PPARG相关性。.①通过TCGA数据库分析以及免疫组化染色验证NUPR1与膀胱癌临床特征之间的关系，证实了NUPR1的表达与膀胱癌的临床分期呈正相关，高级别膀胱癌中NUPR1的表达高于低级别膀胱癌；②使用生信分析NUPR1在膀胱癌不同分期中的预后，证实了晚期膀胱癌的NUPR1高表达的患者相较于早期患者的预后更差；③通过GSEA发现NUPR1与膀胱癌相关。此外，PPAR信号通路也与NUPR1的高表达相关；④通过STRING数据库，发现共有30余种蛋白质与NUPR1有相关性，包括PPARG；⑤通过生信分析，证实了NUPR1的表达与PPAR信号通路相关基因PPARG呈负相关，与PPARA与PPARD无关。