骨髓浸润性T细胞中FOXC1 H446HG突变介导Treg分化在儿童急性淋巴细胞白血病发生中机制研究

Childhood acute Lymphoblastic Leukemia (ALL) is the most common cancer in children. Immunosuppressive microenvironment especially regulatory T cells (Treg) infiltration has been documented highly associated with ALL. This present project based on genetic factors, aiming to investigate the potential mutation involved in the immunosuppressive microenvironment of ALL. High throughput sequencing screening identified that FOXC1 H446HG was participating in the differentiation of Treg in marrow. Verification will be carried out based on amount of clinical samplers. The role of FOXC1 H446HG in infiltrating immunocytes on development of children ALL will be explored by a serial of experiments including in vitro co-culture of inflammatory and ALL cells, in vivo tumor growth induced mice tumorigenesis models. Through a bioinformatics and protein domain analysis, the detailed function of FOXC1 H446HG on FOXC1 and associated KLFs signaling targeting Treg differentiation will be further examined in T cells extracted from marrow. The outcome of this project will lay the theoretical foundation for a better interpretation of inflammation related children ALL pathogenesis and may serve as a potential target of immunotherapy in the future.

儿童急性淋巴细胞白血病（Childhood Acute Lymphoblastic Leukemia, ALL）是儿童时期最常见的恶性肿瘤，免疫抑制微环境特别是骨髓Treg异常分化已被证实与儿童ALL发生发展密切相关。本研究从遗传学角度出发，探索其介导的免疫微环境改变在儿童ALL发病过程中重要作用。通过二代测序技术，筛选出与儿童ALL骨髓浸润性Treg分化密切相关的FOXC1 H446HG突变，并结合大规模临床样本验证其在人群中的分布；同时，基于体外实验探索该突变对Treg分化的影响。通过体外炎、癌细胞共培养，体内肿瘤生长小鼠模型，分析该突变在免疫细胞中异常激活时对白血病作用机制。结合生物学信息学技术/蛋白功能域分析，在骨髓原代T细胞中探索该突变对FOXC1及其介导的KLFs通路在Treg分化中的调控机制。本课题旨在从免疫微环境角度诠释白血病的发生，为后期进行免疫治疗奠定理论基础。

背景：儿童急性淋巴细胞白血病是儿童最常见的恶性血液病。遗传因素及免疫微环境在白血病的发生发展过程中扮演了非常复杂的角色。目前，关于遗传改变介导免疫微环境变化在儿童ALL发生发展中的研究引起人们关注。本文通过生物信息学探索发现，FOXC1 H446HG突变与儿童ALL骨髓浸润性Treg分化密切相关。本文旨在探索骨髓原代T细胞中该突变对FOXC1及其介导的KLFs通路在Treg分化中的调控机制。.方法：扩大病例，并用Sanger法鉴定FOXC1 H446HG基因型，采用qRT-PCR方法检测野生型及突变型患者骨髓FOXC1 mRNA的表达水平；流式细胞术检测ALL患者骨髓原代Naïve T细胞亚群的分布并进行各亚群定向诱导分化；通过Transwell共培养后，采用CCK8及流式细胞术检测ALL细胞增殖、细胞凋亡、细胞周期；NOD/SCID在体小鼠研究FOXC1 H446HG对白血病细胞生长影响；凝胶迁移实验（Electrophoretic Mobility Shift Assay，EMSA）及荧光素酶报告基因实验鉴定FOXC1 H446HG突变在Treg分化过程具体机制。.结果：运用高通量芯片检测及大样本人群验证FOXC1 H446HG突变在ALL患儿中表达明显下调。体外CCK8、流式细胞术检测发现FOXC1 H446HG突变抑制Treg细胞分化，同时抑制白血病细胞的增殖。与体外实验相一致，在动物水平研究中发现FOXC1 H446HG突变型较野生组瘤体体积积明显减小。通过Real-time PCR在瘤组织中检测野生型及突变型中FOXC1及FOXP3的表达差异，结果显示H446HG突变型FOXC1及FOXP3的表达水平明显降低。.结论：FOXC1 H446HG突变在转录水平抑制介导FOXC1低表达，调控KLF10导致Treg分化明显减少，进而降低Treg免疫抑制作用，抑制ALL白血病细胞增殖参与儿童急性白血病发生发展。