BEST4作为Notch2-Hes4轴的关键靶分子通过拮抗STAT3二聚化逆转EMT抑制结直肠癌转移的作用和机制研究

卵黄状黄斑病蛋白4（BEST4）是新发现的肠上皮吸收细胞的分子标志之一。申请人前期研究发现：BEST4在结直肠癌组织中表达降低，与癌转移密切相关；体外实验表明过表达BEST4可抑制结直肠癌细胞的增殖，侵袭及迁移，下调EMT相关因子表达；Notch2和Hes4上调BEST4的表达且Hes4可增强BEST4启动子活性；此外还发现BEST4可与STAT3结合，减少STAT3入细胞核发挥转录活性。由此我们推测：BEST4可能作为Notch2-Hes4轴下游靶分子通过拮抗STAT3二聚化，抑制其转录活性，从而逆转癌细胞EMT表型和抑制结直肠癌的转移。本研究拟结合临床样本，通过细胞实验和动物模型，从分子，细胞，组织和动物整体水平验证假说，明确BEST4与上游Notch2-Hes4信号通路的交互作用并探寻BEST4通过拮抗STAT3二聚化抑制结直肠癌转移的具体机制，为阻遏结直肠癌转移提供重要的基础数据。

Bestrophin 4 (BEST4) was a newly identified marker for colonic absorptive cell. Our previous research found that the expression of BEST4 was decreased in CRC tissues, which was closely related to the metastasis of CRC; in vitro experiments showed that overexpression of BEST4 inhibited the proliferation, invasion, and migration of CRC cell lines; down-regulate the EMT related factors. Stimulation of Notch2 or Hes4, consistently induced their BEST4 gene expression, and the promoter activity of BEST4 was enhanced by Hes4 as determined by dual-luciferase assay. In addition, STAT3 was immunoprecipitated by BEST4; the translocation of STAT3 into the nucleus was reduced and its transcriptional activity was inhibited after BEST4 transfection. Therefore, we hypothesis that BEST4 may act as a key target for Notch2-Hes4 axis which could antagonize the dimerization of STAT3 and suppresses its transcription activity, thus reversing EMT and inhibit the metastasis of CRC. This project intends to combine clinical samples, in vitro and in vivo models to clarify the interaction of BEST4 with its upstream Notch2-Hes4 signaling pathway, and explore the possible mechanism of BEST4 in regulating EMT through antagonizing the dimerization of its downstream target STAT3, and to provide important basic data for inhibiting CRC metastasis.