结直肠癌中B7-H3亚型异常表达的调控机制及功能意义研究

The isoforms of co-stimulatory molecule B7-H3, 2Ig-B7-H3 and 4Ig-B7-H3, have the roles of enhancing anti-tumor immunity and inhibiting tumor immunity, respectively. In colorectal cancer, 2Ig-B7-H3 is downregulated and 4Ig-B7-H3 is upregulated, which leads to tumor immune evasion and is significantly associated with patients’ prognosis. The current study aims to test the scientific hypothesis that a set of microRNAs including miR-34a-5p through regulating the expression of splicing factors and glycosyltransferases lead to upregulation of 4Ig-B7-H3 and downregulation of 2Ig-B7-H3, and consequently result in tumor immune escape. Specifically, we will explore the expression relationship, clinical relevance, regulation mechanisms, glycan structures, and immunological roles of multiple forms of B7-H3, using various detection techniques such as immunohistochemistry, western blotting, real-time PCR, liquid chromatography–tandem mass spectrometry, luciferase reporter assay, flow cytometry, as well as enzyme-linked immunosorbent assay. Furthermore, we will investigate the anti-tumor role of the regulating factors of B7-H3, splicing factors, and glycosyltransferases by using in vitro and in vivo functional experiments. These findings will lead to a better understanding of the regulation mechanisms underlying expression of multiple forms of B7-H3, and facilitate the development of novel specific therapies for the treatment of colorectal cancer.

协同刺激分子B7-H3亚型2Ig-B7-H3和4Ig-B7-H3在肿瘤中分别发挥增强抗肿瘤免疫和抑制肿瘤免疫的作用。结直肠癌高表达4Ig-B7-H3而低表达2Ig-B7-H3，介导肿瘤免疫逃逸，并与患者预后相关。本项目拟围绕“结直肠癌中miR-34a-5p等miRNA通过剪切因子和糖基转移酶分别影响B7-H3亚型表达水平和糖基化水平，导致4Ig-B7-H3表达升高而2Ig-B7-H3表达降低，介导肿瘤免疫逃逸”这一科学问题，采用IHC、WB、qPCR、LC-MS/MS、荧光报告分析系统、流式细胞术、ELISA等方法技术，研究2Ig-B7-H3和4Ig-B7-H3的表达相关性、临床相关性、调控机制、糖链结构及免疫学功能；再结合体内外实验比较研究B7-H3、剪切因子和糖基转移酶等分子调控因子的抗肿瘤活性，以期发现基于调控2Ig-B7-H3和4Ig-B7-H3的有效抗肿瘤治疗靶点或途径。

B7家族协同刺激分子B7-H3在结直肠癌等多种肿瘤中异常高表达，并且与患者预后差显著相关。临床试验结果显示，B7-H3治疗性抗体在多种实体瘤中取得很好的治疗效果。因此，探明B7-H3在肿瘤中异常高表达的分子机制将有助于发现新的治疗靶标。本项目分别从转录后水平和翻译后修饰水平对B7-H3在结直肠癌中异常表达的调控机制进行了深入研究。在转录后水平的研究发现，结直肠癌中异常高表达的miR-34a通过抑制SIRT1，激活NF-κB，上调B7-H3，进而促进TNF-α分泌，介导肿瘤免疫逃逸发生。同时，我们对参与B7-H3剪接的剪接因子进行了筛选。结果发现SRSF3通过与其外显子4和6上的CCCUACU位点结合，导致外显子4和/或5的跳跃，以及内含子3或5的保留。此外，我们还发现岩藻糖基转移酶FUT8通过介导B7-H3核心岩藻糖修饰，保护其不被溶酶体降解，从而增强其稳定性。在此基础上，我们分别以miR-34a和SRSF3为靶点，设计合成了miR-34a突变类似物和SRSF3小分子抑制剂，经体内外活性试验表明，两类化合物均显示出较强的抗肿瘤活性。这些研究结果为发现新的抗肿瘤靶标和药物提供了新的思路和路径。