O型-糖基化异常在结肠癌发生和转移的机制研究

Colorectal cancer is a leading cause of cancer-related death around the world but there are no reliable treatments available. It is very difficult to completely cure colorectal cancer especially for metastatic tumors. Recent evidence suggests that abnormal O-glycosylation plays a pathogenic role in the development and metastasis of colorectal cancer, but its molecular mechanisms remain unclear. Based on our previous data, we are going to deeply investigate: (1) the occurrences of abnormal O-glycosylation in human primary and metastatic colorectal cancer, as well as the relationship between abnormal O-glycosylation and the clinical progress;(2) we first isolate Tn antigen positive tumor cells (which is a specific marker for abnormal O-glycosylation) through laser capture microdissection (LCM) and explore the underlying mechanisms using exome capture sequencing, qRT-PCR, and analysis of the promoter methylation by MassARRAY; (3) we observe if abnormal O-glycosylation affect the migration and invasion of tumor cells, and the capacity of growth, distant metastasis of cancer in vitro and in vivo; (4) we employ FACS to isolate Tn antigen positive tumor cells from primary and metastatic colorectal cancer and use gene expression microarray to analyze the underlying genes and molecular events related to O-glycosylation. Our research will uncover some novel explanations regarding the role of O-glycosylation in the development and metastasis of colorectal cancer and provide some clues for their early diagnosis and targeted therapies in clinical practice.

结直肠癌是常见的恶性肿瘤，根治困难且易复发转移。O-型糖基化异常与结直肠癌的发生发展和远处转移密切相关，但其分子机制尚不清楚。结合我们前期工作的积累，本课题拟研究：1）O-型糖基化异常在人结直肠癌原发灶和转移灶的表达及其与临床转归的关系；2）采用激光捕获显微切割技术从结直肠癌组织中分离Tn抗原阳性肿瘤细胞（O-型糖基化异常特异性标志物），通过外显子捕捉测序、定量PCR和MassARRAY甲基化分析技术明确Tn抗原异常表达的分子机制；3）通过体内外试验比较O-型糖基化异常对肿瘤细胞生长以及肝脏远处转移能力的影响；4）利用流式细胞分选和基因表达微阵列技术探讨结直肠癌原发灶和肝转移灶Tn抗原阳性和阴性细胞基因表达差异和参与的分子事件。本课题将有助于揭示O-型糖基化异常在结直肠癌发生和转移中的地位及分子机制，为结直肠癌的早期诊断、开发新的针对原发病灶和转移病灶的靶向治疗药物提供重要科学线索。

结直肠癌是最常见的消化道恶性肿瘤之一。我们的前期研究表明异常的O-型糖基化过程广泛存在于人结直肠癌中。而O-型糖基化异常在结直肠癌发生发展中的具体作用及分子机制尚不清楚。本项目从多角度探讨了O-型糖基化异常对结直肠癌生物学行为的影响及其可能的分子机制：1）我们应用免疫组化技术检测发现Tn抗原在86%的人结直肠癌原发和转移组织中表达，不在正常的结肠组织中表达。结直肠癌组织中T合酶和分子伴侣Cosmc的表达均有下降。Cosmc基因和T合酶基因启动子区域甲基化水平检测提示Cosmc基因存在过甲基化状态，可能是导致Cosmc表达下降的原因。此外，Tn抗原表达与结直肠癌组织中黏蛋白异常表达相关，异常的黏蛋白如MUC2缺失破坏了消化道黏液屏障从而可促进肿瘤生成。2）构建体外细胞系模型。通过向天然O-型糖基化异常人结直肠癌细胞系LS174T中转染表达分子伴侣Cosmc的载体，恢复其正常O-型糖基化作用，同时应用CRISPR/Cas9技术将天然O-型糖基化正常人结直肠癌细胞系HCT116及SW480细胞中Cosmc基因或T合酶基因进行敲除处理，获得O-型糖基化异常人结直肠癌细胞系。然后通过一系列体内及体外实验证实O-型糖基化异常促进结直肠癌恶性表型。3）对异常O-型糖基化过程诱导EMT发生及其调节机制的研究。我们通过大量细胞分子实验及TCGA数据库分析证实:异常O-型糖基化通过上调H-Ras基因表达水平可诱导上皮-间质转化过程，可能是异常O-型糖基化促进肿瘤恶性行为的重要机制。4）异常O-型糖基化结肠癌细胞合成了大量的未成熟O-型糖基化蛋白如CD44，并由外泌体介导方式释放至胞外及远处受体细胞，从而发挥调节作用。5）发现了肿瘤干细胞标志物DCLK1与消化道肿瘤的发生密切相关。这些研究结果证实异常O-型糖基化与结直肠癌发生与转移密切相关，为人类结直肠癌早诊早治提供了思路与依据。