【作者】 王雪；

【导师】 刘宇博；

【作者基本信息】 大连理工大学 ， 生物工程（专业学位）， 2020， 硕士

【摘要】 乳腺癌是女性发病率最高的恶性肿瘤,也是女性恶性肿瘤死亡首要病因。虽然综合治理改善了患者的预后,然而临床耐药的产生严重制约了乳腺癌患者的治疗效果,也是乳腺癌临床治疗的主要瓶颈之一。揭示乳腺癌耐药的发生发展机制不仅为研究耐药逆转策略提供理论基础,而且对乳腺癌治疗具有实际意义。耐药是肿瘤细胞为适应环境变化所建立的一系列调节方式,除了 P-糖蛋白药物外排转运体的过表达,导致的细胞适应性增强,细胞凋亡功能失调等也会造成耐药。其中,翻译后修饰调控的信号通路改变也是耐药产生的重要原因之一。蛋白质的O-β-N-乙酰葡糖胺(O-GlcNAc)修饰是一种简单的、动态的翻译后修饰,参与众多生理和病理过程的调节,O-GlcNAc转移酶(OGT)和O-GlcNAc糖苷酶(OGA)负责底物蛋白的糖基化添加与消除过程。已有的研究表明,包括药物刺激在内的多种应激反应均有O-GlcNAc修饰蛋白的参与。然而,O-GlcNAc修饰肿瘤细胞耐药的分子机制仍待阐明。硼替佐米(BTZ)是一种用于临床治疗的靶向抗肿瘤药,可诱导细胞凋亡。虽然BTZ在临床上表现出对部分血液肿瘤的治疗效果,但对于其他实体瘤,特别是乳腺癌则疗效不佳。本论文通过蛋白质免疫印迹、RT-qPCR、蛋白质免疫共沉淀等实验手段,分析了乳腺癌细胞在BTZ刺激下,胞内O-GlcNAc修饰的变化,探究了 O-GlcNAc修饰调控乳腺癌细胞BTZ耐药的作用机制。首先,本文研究了内源性BTZ抗性乳腺癌细胞MCF-7、T47D和BTZ敏感的MDA-MB-231细胞中O-GlcNAc修饰的水平。相比BTZ敏感的乳腺癌细胞,内源性耐药MCF-7和T47D细胞中O-GlcNAc修饰在BTZ刺激下动态增加。同时,在BTZ获得性抗性的MDA-MB-231衍生细胞MDA-BR1-3中,随着BTZ抗性越强,O-GlcNAc修饰水平出现升高,提示O-GlcNAc修饰参与了乳腺癌细胞BTZ耐药过程。接下来本文进一步探究了 O-GlcNAc修饰影响BTZ诱导细胞凋亡的分子机制。本文以内源性抗性MCF-7细胞和获得性抗性MDA-BR3细胞为研究对象,发现BTZ诱导的O-GlcNAc修饰上调降低了乳腺癌相关的转录因子FOXA1蛋白质稳定性,进而抑制了乳腺癌细胞的凋亡。在线序列分析结果表明,细胞凋亡重要调控因子Bim启动子区存在FOXA1的结合位点。在BTZ耐药细胞中,FOXA1的O-GlcNAc修饰增强,使FOXA1的蛋白水平和稳定性降低,进而导致下游Bim转录减少,最终阻止细胞凋亡的发生。这些结果证明FOXA1蛋白水平与乳腺癌细胞BTZ抗性及O-GlcNAc修饰呈负相关,O-GlcNAc糖基化调控了 FOXA1依赖的促凋亡蛋白Bim表达。最后,本文使用O-GlcNAc修饰抑制剂L01与BTZ作用于耐药细胞,观察到L01显著增强了 BTZ诱导的细胞凋亡,进一步证明了 O-GlcNAc修饰在乳腺癌细胞耐药产生过程中发挥重要作用。本文的研究结果将O-GlcNAc修饰升高与乳腺癌BTZ耐药性相关联,证明了 O-GlcNAc调控的乳腺癌相关转录因子FOXA1在乳腺癌细胞凋亡失调中发挥功能性作用。BTZ联合O-GlcNAc抑制剂可以克服乳腺癌BTZ耐药,为开发新的乳腺癌治疗策略提供了新思路。更多还原

【Abstract】 Breast cancer is the malignant tumor with the highest incidence in women,and it is the leading cause of death from malignant tumors in women.Although comprehensive management improves the prognosis of patients,the emergence of clinical drug resistance seriously restricts the therapeutic effect of breast cancer patients and is also one of the main bottlenecks in the clinical treatment of breast cancer.Revealing the mechanism of occurrence and development of breast cancer resistance not only provides a theoretical basis for studying drug resistance reversal strategies,but also has practical significance for breast cancer treatmentDrug resistance is a series of regulation methods established by tumor cells to adapt to environmental changes.In addition to the overexpression of efflux transporters of P-glycoprotein drugs,resulting in increased cell adaptability and dysfunction of apoptosis also cause drug resistance.Among them,the change of signaling pathways regulated by post-translational modification is also one of the important reasons for drug resistance.O-β-N-acetylglucosamine(O-GlcNAc)modification of protein is a simple and dynamic post-translational modification,involved in the regulation of many physiological and pathological processes,O-GlcNAc transferase(OGT)and O-GlcNAc glycosidase(OGA)is responsible for the addition and elimination of glycosylation of substrate proteins.Existing studies have shown that O-GlcNAc modified protein is involved in various stress responses including drug stimulation.However,the molecular mechanism of O-GlcNAc modifying tumor cell resistance remains to be elucidated.Bortezomib(BTZ)is a targeted anti-tumor drug used in clinical therapy and can induce apoptosis.Although BTZ has clinically shown a therapeutic effect on some hematological tumors,it is not effective for other solid tumors,especially breast cancer.In this thesis,Western blot,RT-qPCR,protein immunoprecipitation and other experimental methods were used to analyze the changes of intracellular O-GlcNAc modification of breast cancer cells under BTZ stimulation,and to explore the regulation of O-GlcNAc modification to regulate BTZ resistance The mechanism of action of the drug.First,this article studies the level of O-GlcNAc modification in endogenous BTZ-resistant breast cancer cells MCF-7,T47D,and BTZ-sensitive MDA-MB-231 cells.Compared with BTZ-sensitive breast cancer cells,endogenous MCF-7 and T47D cells were dynamically increased under BTZ stimulation under O-GlcNAc modification.At the same time,in BTZ-acquired MDA-MB-231-derived cells MDA-BR1-3,as BTZ resistance is stronger,O-GlcNAc modification levels increase,suggesting that O-GlcNAc modification is involved in breast cancer cells BTZ resistance process.Next,this article further explored the molecular mechanism of O-GlcNAc modification affecting BTZ-induced apoptosis.In this study,endogenous resistant MCF-7 cells and acquired resistant MDA-BR3 cells were used as the research objects.It was found that BTZ-induced up-regulation of O-GlcNAc reduced the stability of breast cancer-related transcription factor FOXA1 protein,which in turn inhibited breast cancer cells.Of apoptosis.The results of online sequence analysis showed that there is a FOXA1 binding site in the Bim promoter region,an important regulator of apoptosis.In BTZ-resistant cells,the O-GlcNAc modification of FOXA1 is enhanced,which reduces the protein level and stability of FOXA1,which leads to the decrease of downstream Bim transcription and ultimately prevents the occurrence of apoptosis.These results prove that FOXA1 protein level is negatively correlated with breast cancer cell BTZ resistance and O-GlcNAc modification.O-GlcNAc glycosylation regulates the expression of FOXA1-dependent pro-apoptotic protein Bim.Finally,this article uses the O-GlcNAc modification inhibitor L01 and BTZ to act on drug-resistant cells.It is observed that L01 significantly enhances BTZ-induced apoptosis,further proving that O-GlcNAc modification plays an important role in the development of breast cancer cell resistance effect.The results of this study correlated the increased O-GlcNAc modification with breast cancer BTZ resistance,demonstrating that O-GlcNAc-regulated breast cancer-related transcription factor FOXA1 plays a functional role in the regulation of apoptosis of breast cancer cells.BTZ combined with O-GlcNAc inhibitors can overcome BTZ resistance in breast cancer and provide new ideas for the development of new breast cancer treatment strategies.更多还原