S100A8核移位通过SETD2介导组蛋白甲基化异常增加炎症相关结直肠癌微卫星不稳定性

Inflammation play an important role in the occurrence and development of colorectal cancer. The applicant found that in the mouse model, the inflammatory factor S100A8 overexpressed among the whole progress of colitis associated colorectal cancer (CAC) developed, and S100A8 translocated to the nuclear in some dysplasia and carcinoma tissues. The patients suffered colorectal cancer with S100A8 overexpressed in the nuclear had the similar clinicopathologic feature to the patients who had highly frequency microsatellite instability (MSI-H) colorectal cancer. In SW480 cell line, SETD2 and H3K36me3 had a lower expression level when S100A8 was overexpressed in the nuclear, and the MSI level also increased in this situation. All these phenomenon indicated that there were some relationships between S100A8 nuclear tanslocation and MSI. In this study, we will first talk about the relationship among S100A8 nuclear translocation, H3K36 abnormal methylation and MSI in the CAC mouse model, human inflammation and carcinoma tissues. Second, we will demonstrate that there is a synergy function between S100A8 nuclear translocation and ZEB1. The two can work together enhance the function to inhibit the transcription of SETD2. The low-expression of SETD2 can lead to the abnormal methylation of H3K36me3, then affected the function of hMSH6, finally result in MSI. The third, we will verified these molecular mechanisms in the original cell lines generated from the human inflammatory intestinal mucosa and colorectal cancer tissues. The completion of this study will be elucidated the abnormal S100A8 nuclear translocation/SETD2/H3K36me3/MSI abnormal signal events play an important role in the development of colitis associated colorectal cancer.

炎症在结直肠癌发生与演进过程中作用关键。申请者发现炎性因子S100A8在“炎-癌”演进小鼠模型中高表达且存在核移位；结直肠癌患者中S100A8核移位者与高频微卫星不稳定（MSI-H）者特征相似；结肠癌细胞中S100A8核移位增加MSI且下调甲基转移酶SETD2和组蛋白H3K36me3水平，组蛋白甲基化修饰缺失可引起DNA错配修复基因定位异常而致其功能不全；因此我们设想炎症相关结直肠癌中，S100A8核移位可协同ZEB1抑制SETD2转录，进而引起H3K36的三甲基化水平异常，导致MSI。本研究拟在动物模型、人体标本中证实S100A8核移位与H3K36甲基化异常和MSI之间的相关性，并在原代培养细胞系及结肠癌细胞中研究S100A8、ZEB1、SETD2、H3K36me3的相互调控关系及与其MSI的关系，阐明S100A8核移位/SETD2/H3K36me3/MSI信号异常参与炎症促发肿瘤机制。

摘要：.本研究结果显示小鼠“炎-癌”序列演进过程中未发现MSH2、MSH6、APC和β-catenin突变。S100A8核内高表达可下调SETD2的表达及组蛋白H3K36me3水平，引起微卫星位点BAT25、BAT26的不稳定性，但对MSH6的表达无影响。在S100A8核内高表达细胞中恢复SETD2的表达可以恢复H3K36me3水平及BAT25位点的稳定性，但同样对MHS6的表达水平无影响。激光共聚焦结果显示，S100A8核移位通过抑制SETD2的表达抑制了MSH6向核小体定位，通过引起MSH6的定位异常影响其功能，进而导致了微卫星不稳定的发生。体内外实验证实S100A8核移位可以促进结肠癌细胞增殖能力及侵袭能力。在S100A8核移位的细胞中恢复SETD2的表达可以部分抑制S100A8核移位引起的促结肠癌细胞增殖和侵袭的作用。生物信息学分析显示SETD2启动子区具有3个RUNX1的结合位点。基于TCGA肿瘤组织数据库和GTEx正常组织数据库进行生物信息学分析，RUNX1在结直肠癌组织中的表达水平显著高于正常结直肠组织。免疫共沉淀结果显示RUNX1可以与S100A8相结合。S100A8核移位的情况下，干扰RUNX1的表达可以恢复结肠癌细胞中SETD2的表达。采用RUNX1抗体进行共沉淀，ChIP-qPCR检测结果显示干扰RUNX1的表达后，其与预测的SETD2启动子区B位点和C位点的结合显著减少，进一步采用S100A8抗体对核蛋白进行沉降，结果显示在SETD2启动子区存在S100A8和RUNX1的结合，干扰RUNX1后，S100A8与RUNX1在SETD2启动子区的结合显著减少。免疫组化结果进一步证实在高频微卫星不稳定的人结直肠癌组织中存在明显的S100A8核内高表达，在此类型结直肠癌组织中SETD2表达显著下调，H3K36me3水平显著降低，同时hMSH6阳性表达主要定位于胞浆。本研究初步证实了在结直肠癌中，S100A8核移位通过与RUNX1协同作用，抑制了SETD2的转录和表达，进而引起H3K36位点甲基化异常，导致MSH6向核小体定位异常，引起后者功能的缺失进而导致微卫星不稳定的发生，促进了结直肠癌的进展。丰富了炎症促进肿瘤发生的发病机制，为结直肠癌的预防、早期诊断及个体化治疗研究提供新的思路及靶点。