Sox2在结直肠癌Ras/Rac1细胞运动信号通路中的作用及其分子机制

Our previous findings indicate that miR-200c and Sox2 reciprocally control their expression through a feedback loop not only modulating Sox2-induced stemness but also mediating proliferation and metastasis in colorectal cancer (CRC). Sox2 was statistically higher in colorectal cancer tissues than that in the normal counterparts. Sox2 overexpression increases colorectal cancer cell proliferation, migration, and invasion. However, the mechanism of Sox2 in promoting colorectal cancer cell proliferation and metastasis remains largely unknown. The most differentially modulated proteins of Sox2 were assayed by silver nitrate staining and mass spectrometry, in which ENO1 was upregulated and confirmed by RT-PCR and Western Blot. ENO1 was overexpressed in colorectal cancer. High-level expression of ENO1 was significantly associated with tumour size, serosal invasion, lymph metastasis, and tumour–node–metastasis in colorectal cancer. The suppression of ENO1 inhibited colorectal cancer cell proliferation, migration, invasion, and the expression of Ras and Rac1. Therefore, we reasoned that ENO1 is a target of Sox2 in driving colorectal cancer cell metastasis through triggering of Ras/Rac1 cell movement signal pathway. Our project will provide novel insights into cancer metastasis, and new way for targetedly suppressing Sox2-related signaling pathway to inhibit metastasis.

前期研究，我们发现miR-200c-Sox2通过负反馈机制调节结直肠癌的干性、生长和转移；Sox2是miR-200c的靶基因，在结直肠癌中表达升高并促进结直肠癌细胞的生长和转移，但其机制尚未明确。ENO1是我们筛选的Sox2下游调控蛋白，在结直肠癌组织和高转移潜能结直肠癌细胞中表达升高，且其表达水平与结直肠癌的大小、浆膜浸润、淋巴结转移以及TNM分期显著相关；瞬时干扰ENO1的表达抑制结直肠癌细胞的增殖、迁移、侵袭及Ras和Rac1的表达。这些结果表明ENO1表达下调可以抑制结直肠癌的转移。因此，我们提出了Sox2调控下游靶分子ENO1并激活Ras/Rac1通路而促进结直肠癌转移的新假说。本研究将探讨Sox2对ENO1、Ras/Rac1信号通路及Rac1下游效应蛋白PAK1、MLC、LIMK1和DLC1的调节机制，以阐明Sox2促进结直肠癌转移的机制，为抗结直肠癌转移治疗提供新的分子靶标。

前期研究，我们发现miR-200c-Sox2负反馈机制调节结直肠癌的的干性、生长和转移。但是，Sox2促进结直肠癌发生发展的机制仍未明确。应用蛋白质质谱分析我们初步发现Sox2的下游调控蛋白ENO1，在结直肠癌细胞中，上调Sox2的表达，ENO1表达升高；沉默Sox2的表达，ENO1表达下降。根据前期研究结果，我们提出了Sox2通过ENO1激活Ras/Rac1细胞运动信号通路而促进结直肠癌转移的假说。因此，我们展开如下研究：（1）、采用免疫组化的方法检测ENO1在结直肠癌组织中的表达，并分析两者的表达与结直肠癌的生长、转移、分期、分化的关系；（2）、采用蛋白质免疫共沉淀（CO-IP）及共定位技术验证Sox2与Oct-1的相互作用；（3）、采用双荧光素酶报告实验和染色质免疫共沉淀（ChIP实验）技术证实Oct-1对ENO1的转录调控；（4）、体内外实验证实ENO1在结直肠癌细胞生长和转移中的作用；（5）、证实Sox2对ENO1、 Ras/Rac1信号通路的调节。我们的研究结果表明：（1） Sox2与Oct-1相互作用并通过Oct-1与ENO1启动子结合而调控ENO1的转录；（2） ENO1在结直肠癌组织和具有高转移潜能的结直肠癌细胞中表达升高，且ENO1的表达水平与结直肠癌的大小、浆膜浸润、淋巴结转移以及 TNM 分期显著相关；（3） Sox2通过ENO1激活Ras/Rac1信号通路而促进结直肠癌的生长和转移。综上所述，我们证实了Sox2与Oct-1相互作用并通过Oct-1与ENO1启动子结合而调控ENO1的转录，ENO1激活Ras/Rac1信号通路而促进结直肠癌细胞的转移。