阿司匹林单独与联合用药抗乳腺肿瘤及延缓耐药分子机制研究

As a hundred years old drug, aspirin has an extremely magical property. It can affect various physiological activities of animals and even plants, it is widely used in every clinical fields. A flurry of clinical epidemiological data showed that aspirin protected from cancers and reduced the rate of mortality . The exact mechanism by which aspirin inhibits tumors has not been fully elucidated. At present, tumor drug resistance is the leading cause of tumor low cure rate and the main reason for the tumor metastasis, drug combination is the main method of clinical anti-tumor therapy. Based on the existing research results and the unsolved scientific issues, this project decided to take the antitumor effect of aspirin as a breakthrough to expound the negative effect of aspirin to breast neoplasms and the molecular mechanism of enhanced sensitivity to tamoxifen and cisplatin. At the same time, we establish drug resistance cell lines to tamoxifen and cisplatin, we also use the oncomolecularbiology, cytobiology and other scientific research methods to screen differentially expressed genes between parental cells and drug-resistant cell, to explore the correlate between differentially expressed gene and the ability of invasion, metastasis, proliferation and formation of tumor cells, clarify the significance of differential expressed gene as a biological target of anti-tumor and integrate the tumor microenvironment, the project fundamentally represented related role which aspirin played in the development and progression of mammary gland tumors in order to reveal the occurrence and metastasis mechanism of mammary gland tumors and provide theoretical foundation for the prevention and control measures.

阿司匹林作为百年老药，它能影响动物甚至是植物体内多种生理活动，被广泛应用于临床各领域。多项临床流行病学研究数据表明，阿司匹林可预防癌症的发生及降低癌症的死亡率。阿司匹林抑制肿瘤具体机制尚未完全阐明。肿瘤耐药是目前导致肿瘤治愈率不高及转移的主要原因，联合用药是目前临床抗肿瘤治疗主要方法。本项目根据现有的研究成果及待解决的科学问题，决定以阿司匹林抗肿瘤作用为突破口，阐述阿司匹林抗乳腺肿瘤作用及提高抗癌药物他莫昔芬、顺铂敏感性的分子机制。同时，建立耐他莫昔芬、顺铂乳腺肿瘤细胞系，利用肿瘤分子生物学和细胞生物学等科研手段，筛选亲本细胞与耐药细胞差异基因，探究差异基因与肿瘤细胞侵袭、转移、增值和成瘤能力之间的相互关系，阐明差异基因作为抗肿瘤的生物学靶点的意义并结合肿瘤微环境，从根本上阐述阿司匹林在乳腺肿瘤发生发展过程中的关联作用，为揭示乳腺肿瘤的发生及转移机理和为乳腺肿瘤有效的防控提供理论依据。

犬乳腺肿瘤（CMGT）作为常见的肿瘤，如同女性乳腺肿瘤常常发生恶性并伴有转移，对犬的健康造成严重危害。目前手术和化学疗法是乳腺癌治疗的主要手段。但大多数化疗药物具有细胞毒性作用和不良反应，同时由于药物耐药性产生大大增加了患者的复发率和死亡率。因此，探究耐药机理，克服耐药产生及增强机体对药物的敏感性对于治疗乳腺癌具有重要意义。.阿司匹林作为经典的非甾类抗炎药，能影响动物甚至是植物体内多种生理活动，具有解热、镇痛、抗风湿、防治心脑血管疾病等功效，被广泛应用于临床各领域。近年来，多项临床流行病学研究数据表明，阿司匹林的摄入量与多种癌症的预防及死亡率相关。相对于阿司匹林在结直肠癌及胃肠相关的肿瘤中的研究，关于阿司匹林在乳腺肿瘤中的研究较少。而寻找有效的化疗药物及早期诊断和分子靶向治疗是提高患者生存率的关键。.综上，本研究以犬乳腺肿瘤细胞（CMGTs）为研究对象，首先通过CCK8、Transwell、克隆、微乳球实验等，探究阿司匹林在乳腺肿瘤中发生发展及耐药中作用，结果显示阿司匹林通过经典Wnt和PI3K信号通路以浓度依赖抑制CMGTs增殖、迁移、侵袭和克隆能力。同时阿司匹林可以增强顺铂、他莫昔芬药物抗肿瘤作用。然后，通过浓度递增和大浓度药物维持的方法构建三株耐药细胞系，CHMpTAM、CHMmTAM和CHMpCIS，表型实验表明耐药细胞显示更强的增殖、迁移能力。高通量测序技术检测了差异表达的lncRNA、miRNA和mRNA分子，通过生物信息预测、双荧光素酶、核质实验等阐明其耐药药性产生主要与lnc-42060-miR204-SOX4轴激活相关。其次，通过药敏实验表明阿司匹林可以恢复耐药细胞药物敏感性。最后通过生化功能、病理组织学、qRT-PCR和Western blot等实验表明阿司匹林不仅具有抗肿瘤效果，还可以通过AMPK-PGC-1α通路从抗炎、抗凋亡、抗氧化缓解顺铂所致小鼠急性肾损伤。