CD44-TAF1-DPYD通路调控胃癌肿瘤干细胞对5-氟尿嘧啶敏感性的机制研究

Cancer stem cells are thought as the important factor in chemotherapy resistance of tumors. However, it is still not clear that how the sensitivity is and what is the regulating mechanism of gastric cancer stem cells to 5-fluorouracil (5-Fu) as the common drug of chemotherapy of gastric cancer. In the previous researches, our team has captured the primary gastric cancer stem cells from gastric cancer patients and found that CD44 was the specific marker of gastric cancer stem cells (Cell Res. 2012). After co-cultured with 5-Fu, the ratio of CD44 positive cells of gastric cancer stem cells was increased. When knockdown of CD44 expression, the sensitivity to 5-Fu of gastric cancer stem cells was increased, and the expressions of transcriptor TAF1 and the gene DPYD which is closely associated with the metabolism of 5-Fu were down regulated. Based on these results, we propose that whether CD44 could mediate the sensitivity to 5-Fu of gastric cancer stem cells through influencing the activity of TAF1 and regulating the expression of DPYD? This research aims to illustrate the mechanism of CD44-TAF1-DPYD pathway in regulating the sensitivity to 5-Fu of gastric cancer stem cells and the clinical significance of this pathway in gastric cancer tissues through the experiments like CRISPR/CAS9, co-immunoprecipitation, immunofluorescence, quantitative PCR, Western Blot, animal model, et al. This research might provide the experiment evidence in screening the therapeutic targets against 5-Fu resistance of gastric cancer.

肿瘤干细胞被认为是肿瘤化疗耐药的重要因素，但目前胃癌肿瘤干细胞对胃癌常用化疗药物5-氟尿嘧啶（5-Fu）的敏感性及其调控机制尚不清楚。团队前期已捕获人原代胃癌肿瘤干细胞，并发现CD44是其特征性标志物（Cell Res. 2012）；与5-Fu共培养后，胃癌肿瘤干细胞中CD44阳性细胞率上升；干扰CD44表达后，胃癌肿瘤干细胞对5-Fu的敏感性提高，且转录因子TAF1、与5-Fu代谢密切相关的DPYD表达下调。据此，我们提出CD44是否可能通过影响TAF1的活性，调控DPYD表达，介导胃癌肿瘤干细胞对5-Fu的敏感性？本课题拟通过CRISPR/CAS9、免疫共沉淀、免疫荧光、定量PCR、Western Blot、动物模型等实验技术，阐明CD44-TAF1-DPYD通路调控胃癌肿瘤干细胞对5-Fu敏感性的分子机制，论证其在胃癌组织中的临床意义，为筛选胃癌对5-Fu治疗耐药的靶点提供实验依据。

胃癌肿瘤干细胞被认为是胃癌发生发展及治疗耐药的重要因素。CD44是胃癌肿瘤干细胞的特征性标志物，并调控其多种生物学行为。目前针对CD44细胞内域（CD44 intracellular domain，CD44ICD）的研究还不多，其调控胃癌肿瘤干细胞生物学行为的分子机制不清楚。本项目主要研究了CD44ICD对胃癌肿瘤干细胞生物学行为的影响及其分子调控机制，同时在胃癌组织中检测CD44和KIAA1199的表达水平，并分析其临床意义。本项目发现干扰CD44的表达可增加胃癌肿瘤干细胞对5-氟尿嘧啶的敏感性，但CD44ICD过表达对5-氟尿嘧啶化疗敏感性无显著影响。与对照组相比，CD44ICD过表达组胃癌肿瘤干细胞的体外增殖能力、克隆形成能力和体内皮下成瘤能力更强，但迁移能力降低。进一步通过测序及生物信息学分析发现KIAA1199可能是CD44ICD的下游分子。CD44ICD过表达后，KIAA1199的表达降低。敲低KIAA1199后，胃癌肿瘤干细胞的体外增殖能力、克隆形成能力显著增强，体内皮下成瘤能力也有所提高，但迁移能力显著下降，与CD44ICD过表达效果相似。而KIAA1199过表达组胃癌肿瘤干细胞较对照组细胞的迁移能力明显增强，但体内成瘤能力下降。在临床样本中，研究发现CD44与患者不良预后显著相关，在5-氟尿嘧啶化疗的人群中，CD44高表达患者预后更差。KIAA1199低表达与淋巴结转移显著相关，且与不良预后有一定相关。本项目研究发现CD44ICD可能通过KIAA1199介导促进胃癌肿瘤干细胞的增殖能力、克隆形成能力和体内成瘤能力，但抑制胃癌肿瘤干细胞的迁移能力。胃癌组织中CD44高表达与不良预后相关，而KIAA1199低表达与胃癌淋巴结转移有关，但其临床意义需要更大样本研究予以验证。本项目研究结果为进一步理解胃癌肿瘤干细胞的生物学特性提供了实验依据，特别是发现在CD44ICD-KIAA1199信号轴可调控胃癌肿瘤干细胞增殖、成瘤、迁移等生物学特性，为靶向胃癌肿瘤干细胞的治疗提供潜在靶点。