CRM1介导的BATF2出核效应促结肠癌增殖的作用及机制研究

Tumor-suppressor gene inactivation is closely related to the genesis and development of colon cancer, of which nuclear translocation is an important way. The tumor-suppressor gene BATF2 suppresses tumor by inhibiting AP-1 in the nucleus. Its anti-cancer effect is inhibited in colon cancer, but the mechanism remains elusive. This research finds different nuclear and cytoplasmic localization of BATF2, and that its nuclear expression positively correlates with prognosis of colon cancer patients. Bioinformatics reveals BATF2 amino acid sequence contains nuclear export signal and BATF2 could directly bind a nuclear transporter CRM1. Base on these preliminary experiments, we speculate BATF2 directly binds CRM1 via its NES, resulting in BATF2 nuclear export and AP-1 activation, which leads to cancer proliferation and poor prognosis of colon cancer patients. With NES mutation and CRM1 inhibition, we would demonstrate the molecular mechanism of BATF2 nuclear export. Further, xenograft mice would be constructed using colon cancer cells transfected with NES mutated BATF2 plasmid, to illuminate the effect of cytoplasmic BATF2 on colon cancer proliferation. This study for the first time investigated the abnormal subcellular localization of BATF2, the molecular mechanism and the biological significance, which could contribute to develop novel therapeutic target for colon cancer.

抑癌基因失活与结肠癌发生发展密切相关，而核转位是其失活的重要途径。抑癌基因BATF2通过在胞核内抑制AP-1发挥抑癌作用，但在肿瘤中其抑癌作用降低，机制不清。本课题首次发现BATF2在结肠癌胞核、胞质存在不同定位，且其核表达与结肠癌患者预后呈正相关。鉴于生物信息学分析BATF2含有出核信号NES，能与CRM1直接结合。我们推测：BATF2通过NES结合CRM1而出核，导致AP-1活性增强而促进结肠癌增殖，影响患者预后。本项目拟通过突变出核信号NES及抑制CRM1表达，解析BATF2核转位的分子机制。进一步利用NES突变的结肠癌细胞构建荷瘤鼠模型，阐明BATF2出核后对结肠癌增殖的影响。本研究首次对抑癌基因BATF2异常亚细胞定位的现象、机制及生物学意义进行探讨，有望为探索新型结肠癌治疗靶点提供理论依据。

目的：包括我们在内的新研究表明，BATF2是一种肿瘤抑制因子，但其亚细胞定位和生物学功能尚不明确。本研究旨在探讨BATF2在结直肠癌(crc)中的定位作用和机制。.方法：我们首先在肿瘤样本和细胞系中鉴定了BATF2的亚细胞定位。采用共免疫共沉淀(co-ip)、定量无标记液相色谱-串联质谱(lc-ms-ms/ms)蛋白质组学分析、western印迹法和免疫荧光法筛选与BATF2结合的候选蛋白。利用重组质粒、点突变和小干扰rna(sirna)，明确了BATF2与CRM1之间的结合位点。在CRC患者、细胞系或异种移植裸鼠中检测其信号通路和临床相关性。 .结果：BATF2主要定位于正常细胞的细胞核，而是在肿瘤细胞的细胞质中。核BATF2的表达与CRC患者的预后呈正相关。然后，我们在BATF2中确定了NES区域(lxxxxxxlxl)，通过该区域BATF2与CRM1结合并运输出细胞核，最终促进CRC的生长。Ap-1/cyclind1/prb信号通路参与了这一过程，可通过BATF2中NES的突变或抑制CRM1来改善。此外，CRM1的表达与核BATF2的表达和CRC的预后呈负相关。.结论：这些发现为BATF2的亚细胞定位和生物学功能提供了新的思路。通过突变BATF2的细胞质区域或抑制CRM1的表达来抑制其细胞质积累，可能是抑制CRC进展的潜在治疗策略。