TWF2通过细胞外囊泡途径影响肾癌细胞干性、肿瘤侵袭转移及靶向药物耐药的分子机制研究

Our previous studies and external databases showed that TWF2 gene methylation and mRNA expression were significantly correlated with the prognosis of renal cell carcinoma, but the role and molecular mechanism of TWF2 as a new biomarker in cancer has not been reported. In this project, we intend to detect TWF2 protein expression in RCC tissues by immunohistochemical method, and evaluate the relationship between TWF2 expression and tumor metastasis/recurrence, targeted treatment resistance and patients’ survival in ccRCC. We then detect the changes of self-renewal ability, stemness markers and related genes expression, invasion and metastasis ability, and the sensitivity to sunitinib after TWF2 gene overexpression and knockout in ccRCC cell lines, to confirm the role of TWF2 on RCC tumor stemness, cell invasion and metastasis, and targeted drug resistance. Finally, we intend to use plasmid transfection, immunoprecipitation, point mutation and sequence deletion, a series of experiments to explore the molecular biology of TWF2 promotes Hedgehog protein by extracellular vesicle pathway from the cell membrane to release, enhance the activation of the Hedgehog signaling pathway in the tumor microenvironment. We hope to elucidate the molecular mechanism by which TWF2 promotes targeted therapy resistance and the metastasis of RCC, to provide the experimental basis for understanding the molecular mechanism underlying the progression of RCC and the selection of therapy targets in RCC.

我们前期研究及外部数据库显示TWF2基因甲基化和mRNA表达与肾癌患者预后显著相关，但TWF2作为新标志物在肿瘤中的作用及分子机制尚无研究报道。本项目拟应用免疫组化在临床肾癌组织标本中检测TWF2表达水平，评估TWF2表达与肾癌患者转移复发、靶向治疗耐药和患者生存的关系；检测TWF2基因过表达和敲除后肾癌细胞株自我更新能力、干性标志物及相关因子表达情况、体内外侵袭转移能力及对靶向药物舒尼替尼敏感性的变化，明确TWF2对肾癌细胞干性、侵袭转移及靶向药物治疗耐药的影响；进一步通过质粒转染、免疫共沉淀和点突变序列缺失等一系列分子生物学实验探索TWF2促进Hedgehog蛋白通过细胞外囊泡途径从肾癌细胞膜的释放，在肾癌肿瘤微环境中增强激活Hedgehog信号通路的调控机制。期望能阐明TWF2促进肾癌侵袭转移和靶向药物耐药的分子调控机制，为深入理解肾癌恶性进展的分子机制及治疗靶点的选择提供实验基础。

通过肾癌DNA甲基化芯片筛选出差异甲基化位点，并成功通过LASSO模型建立肾癌DNA甲基化预后模型。该模型包括了五个基因的CpG甲基化位点：RIN1、EHBP1L1、PITX1、TWF2和FOXE3，提示这5个基因可能在肾透明细胞癌的恶性进展中发挥关键作用。本项目明确TWF2表达升高促进肾癌病人转移复发，降低肾癌舒尼替尼靶向治疗敏感性，缩短肾癌患者的无病生存期和总生存期。阐明TWF2促进肾癌侵袭转移的分子调控机制，利用免疫共沉淀筛选及高分辨率液相色谱-质谱验证TWF2蛋白与下游靶点蛋白YAP相结合，通过多种体外细胞实验、体内动物实验、生物信息学方法证明了TWF2通过与YAP结合促进肾癌细胞的增殖迁移侵袭转移、靶向药物治疗耐药并影响肾癌细胞的干性。揭示了TWF2在肾癌肿瘤干性通路中结合靶点蛋白的新型调控机制。并应用单核苷酸多态性(single nucleotide polymorphism，SNP)芯片、DNA甲基化芯片、转录组测序、circRNA芯片等技术，对肾癌复发转移、靶向药物及免疫检查点抑制剂治疗敏感性相关分子标志物包括SNP、DNA甲基化、 mRNA以及各种非编码RNA（如miRNA、lncRNA和circRNA等）分子标志物的筛选，并构建多分子预测模型，进一步构建基因组学-病例组学融合的多模态分子预测模型用于指导局限性肾癌术后辅助治疗及晚期肾癌的诊疗决策；完成筛选出的相关分子标志物（例如lncRNA IGFL2-AS1、circSDHC、EHBP1L1等）在肾癌侵袭转移、肾癌免疫微环境以及肾癌靶向及免疫治疗耐药中的分子作用机制研究。