STON2/MUC1通路调控卵巢癌类干细胞干性特征的机制研究

Cancer stem cells possess the ability of self-renewal, unlimited proliferation，and non-sensitivity to physical and chemical factors, which is regarded to be a cause of ovarian cancer recurrence, but the mechanism is currently unclear. We found that the expression of STON2 was significantly lower in ovarian cancer-stem like cells by LC-MS/MS-based label free quantitative proteomics approach and Western Blot validation. And its expression in low differentiated ovarian cancer tissue is lower than in the middle and medium high differentiated ovarian cancer tissue. Our previous primary study showed that a facilitation of cancer stem-like cells properties by STON2 knock-down, with MUC1 high expression. In this study proposal, we plan to observe the effect of regulating STON2 expression on the ovarian cancer stem-like cells properties by serum-free culture and gene modulation. To identify that MUC1 participates directly in the process of STON2 mediating the regulation of the ovarian cancer stem-like cells properties by RNA-seq, IP, Immunofluorescence localization, and gene modulation. Finally, To verify the function of STON2 in ovarian cancer by CRISPR/Cas9, animal models and clinical samples. The aim of our study is to expound a novel mechanism that STON2 regulating the ovarian cancer stem-like cells properties and find out a potential strategy for ovarian cancer therapy.

肿瘤干细胞具有自我更新和无限增殖能力，并对外界理化因子的杀伤作用不敏感，被认为是癌症复发的原因之一，但其机制迄今尚不明确。我们通过定量蛋白质组学发现，STON2在3AO卵巢癌类干细胞的表达显著降低，且在低分化卵巢癌组织中表达明显低于中、中高分化卵巢癌组织，下调STON2表达能促进卵巢癌类干细胞干性活性，同时MUC1表达升高。本项目在已有工作基础上拟采用无血清悬浮培养、基因调控等手段观察调控STON2对卵巢癌类干细胞干性特征的影响，并通过RNA-seq，信息学分析、免疫共沉淀、免疫荧光共定位、反向调控等技术证实干性相关分子MUC1直接参与STON2对干性的调控，最后利用CRISPR/ Cas9 技术、动物模型和临床标本验证STON2的功能，从而阐明STON2调控卵巢癌类干细胞干性水平的分子机理，并揭示靶向调控STON2具有的潜在应用价值，为卵巢癌治疗提供新的科学证据。

背景：肿瘤干细胞具有自我更新、分化能力及潜在的致瘤能力，被认为是癌症复发的原因之一。然而，肿瘤干细胞维持其干性的机制仍不清楚。.方法：在本研究中，卵巢癌干细胞样细胞由卵巢癌细胞3AO和Caov3在无血清培养基中富集。使用液相色谱质谱无标记定量蛋白质组学比较卵巢癌干细胞样细胞亚群和亲代细胞之间的差异表达蛋白质；进行自我更新成球能力测定、流式细胞术、定量实时聚合酶链反应、蛋白质印迹和裸鼠皮下成瘤实验评估干性特征；全基因组测序和焦磷酸测序用于揭示STON2负调节卵巢癌干细胞样细胞干性特征的机制。.结果：在74种差异表达的蛋白质中，我们发现STON2在卵巢癌干细胞样细胞中低表达。此外，我们揭示STON2负向调节卵巢癌细胞的干细胞样特性，主要表现为自我更新成球能力、CD44+CD24-比率以及肿瘤干细胞干性标志物和上皮间质转化EMT相关标志物。体内实验发现STON2敲低促进裸鼠皮下成瘤。进一步机制研究发现下游靶点MUC1，随着STON2的下调而上调。MUC1启动子区域中DNA甲基转移酶1(DNMT1)表达和甲基化的降低与随后升高的MUC1表达相关，DNMT1敲低同时提高了MUC1表达，这种STON2-DNMT1/MUC1通路的功能意义得到了STON2过表达抑制MUC1诱导的卵巢癌干细胞自我更新成球能力的观察结果的支持。我们在卵巢癌石蜡切片标本中检测STON2和MUC1的配对表达，揭示了STON2表达的预后价值高度依赖于MUC1表达。.结论：我们的结果表明STON2可能通过DNMT1-MUC1介导的表观遗传修饰负调节卵巢癌细胞的干性，为探索卵巢癌治疗新靶点提供科学证据。.