Wnt信号通路新调控分子BRG1在结肠癌血管生成中的作用及其机制研究

Angiogenesis is essential for tumor growth，invasion and metastasis. In our previous study, the positive correlation between chromatin-remodeling protein BRG1 expression and microvessel density was observed in colon carcinoma specimens. We considered BRG1 as a new potential regulator of angiogenesis in cancer. Aberrant activation of Wnt signaling plays an important role in tuomor angiogenesis. BRG1 and β-catenin co-activates Wnt signaling pathway. Our preliminary data demonstrated that BRG1 enhanced the transcriptional activities of angiogenesis-related gene mediated by frizzled 7 (FZD7). On these grounds, we infered that BRG1 activated Wnt signaling at two levels: interacting with β-catenin and upregulating FZD7. In this study, we will firstly investigate the pathological significance of BRG1 in proliferation，invasion and metastasis of colon carcinoma by RNA interference, gene transfection and small living animal imaging technology. Then we will explore the relationship between BRG1 andβ-catenin, FZD7 using co-immunoprecipitation, chromatin immunoprecitation, sequencing and tissue microarray. Finally, we will study the significance of stimulator and inhibitor of Wnt pathway, β-catenin siRNA in colon carcinoma angiogenesis mediated by BRG1. The aim of this study is to clarify a novel transcriptional regulation mechanism in colon carcinoma angiogenesis and provide a new target for diagnosis and therapy of colon carcinoma.

血管生成在肿瘤生长、侵袭和转移过程中起重要作用。我们发现结肠癌中染色质重构蛋白BRG1表达水平与微血管密度呈正相关，是肿瘤血管生成潜在新调控分子。文献报道Wnt信号通路活性影响肿瘤血管生成；BRG1与β-catenin形成复合物协同激活Wnt通路。课题组前期实验提示BRG1上调卷曲蛋白7（FZD7）受体从而增强血管生成相关基因转录。据此推测BRG1通过与β-catenin直接相互作用和上调FZD7在两水平上激活Wnt通路起促结肠癌血管生成作用。本项目应用RNA干扰、基因转染和小动物活体成像技术研究BRG1对结肠癌增殖、侵袭、转移和血管生成的影响；免疫共沉淀、染色质免疫沉淀、测序和组织芯片技术明确BRG1与β-catenin、FZD7的关系；并探讨WNT通路激活剂、抑制剂和β-catenin敲除对BRG1促血管生成作用的影响。旨在阐明转录水平结肠癌血管生成新调控机制，为临床诊疗提供新靶点。

BRG1作为染色质重构蛋白对基因组转录进行调控，其在细胞内行使功能是通过下游的靶基因表达改变实现。我们应用BRG1 siRNA稳转细胞株和基因芯片技术，检测BRG1敲除前后，结肠癌细胞株中通路和基因表达改变。通过应用芯片和生物信息学分析我们证实了BRG1对结肠癌细胞中Wnt通路的调控；应用免疫组织化学和组织芯片技术证实了BRG1参与结肠癌发生发展；并且在体外、体内水平分别证明了BRG1在结肠癌细胞株增殖及侵袭中的功能。为了能够对BRG1的调控功能有更全面的了解，借助全基因组表达谱芯片，我们分析了BRG1表达变化引起的基因表达变化。通过对表达差异显著的1563个基因进行下游靶标预测以及经过RT-PCR验证，我们初步推断WNT3A可能是BRG1的下游靶基因。在细胞水平及病理标本中，我们发现WNT3A的表达同BRG1表达呈正相关，初步证明了BRG1与WNT3A的表达密切相关。经过通路分析，我们发现BRG1对Wnt/β-catenin通路的多个基因均有正向调控作用，并且通过进一步的实验证明了BRG1通过上调WNT3A表达激活Wnt/β-catenin通路，调控肠癌的细胞增殖、侵袭。课题组在进行课题前期的数据分析时，发现eIF3E的表达在结肠癌组织中显著增高。结合临床资料分析及细胞功能实验研究发现：eIF3E的表达对结肠癌细胞株的增殖侵袭能力及凋亡有很大影响，并与结肠癌患者不良预后密切相关。通过阅读文献，我们发现BRG1通过改变CD44基因启动子甲基化程度来调控CD44的表达，因此我们在前期研究的基础上进一步探究了结肠癌细胞中BRG1同CD44的表达关系，发现在结肠癌细胞中BRG1的表达同CD44呈正相关。项目研究成果在International Journal of Clinical and Experimental Pathology、Disease Markers、Tumor Biology、Oncotarget等期刊发表。