水通道蛋白3通过增强自噬促进胃癌对顺铂耐药

发布时间：2018-09-01 09:34

【摘要】：背景胃癌(GastricCancer,GC)是我国最常见的恶性肿瘤之一,其发病率居消化道肿瘤之首,死亡率列全部肿瘤的2～3位,是严重威胁我国人民生命健康的疾病之一。目前胃癌治疗方法主要包括根治性手术治疗、化学药物治疗(简称"化疗")以及分子靶向治疗等。在我国,绝大部分胃癌患者确诊时多为进展期,而进展期胃癌患者必须有赖于新辅助化疗或术后辅助化疗以改善其长期生存,而化疗耐药的产生常导致化疗失败,严重影响患者预后。顺铂(cisplatin,cDDP)作为治疗胃癌的一线化疗药物之一,其耐药性也在不断上升。因此,深入研究胃癌对顺铂化疗的耐药机制,降低胃癌对顺铂的耐药性,是提高胃癌疗效的重要策略之一。水通道蛋白3(aquaporin3,AQP3)是水通道蛋白家族(aquaporins,AQPs)成员之一,具有介导渗透压依赖性跨生物膜水转运作用,对甘油等小分子物质亦具有通透性。我们前期研究表明AQP3在胃癌组织中过表达,AQP3在胃癌的肿瘤发生发展过程中可能发挥重要作用,AQP3的上调通过促进上皮-间质转化(EMT)和胃癌细胞的干细胞干性促进GC细胞的侵袭和转移。但是,AQP3在胃癌对化疗药物耐药中的作用尚不清楚。自噬(autophagy)是真核细胞胞质内的大分子物质和细胞器被运送至溶酶体隔离并降解的过程,从而实现细胞自身的代谢需要和某些细胞器的更新。自噬是生理条件和病理条件下普遍存在的现象,不仅参与维持细胞稳态,同时还与肿瘤的发生发展以及肿瘤耐药密切相关。深入了解AQP3在促进胃癌细胞自噬中的作用,有助于阐明胃癌化疗耐药的机制,进一步明确AQP3在胃癌化疗耐药中的作用,将为AQP3作为胃癌治疗的靶点提供理论依据和实验基础。方法1.选取三种胃癌细胞株,即MGC803、SGC7901和AGS细胞,分别用慢病毒转染,使MGC803和SGC7901低表达AQP3(标记为MGC803-AQP3low,SGC7901-AQP3low),AGS 高表达 AQP3(标记为 AGS-AQP3high)。2.采用CCK-8细胞毒性试验检测不同AQP3表达水平的细胞对cDDP的敏感性以及对cDDP和氯喹(chloroquine,CQ)联合用药的敏感性。3.采用Western blot、激光共聚焦等方法检测AQP3与自噬相关蛋白的表达。结果1.胃癌细胞中AQP3的表达水平与其对化疗药物顺铂的敏感性显著相关:低表达 AQP3 后,MGC803-AQP3low及 SGC7901-AQ3low对 cDDP 敏感性增强;高表达AQP3后,AGS-AQP3high对cDDP敏感性降低。2.胃癌细胞中AQP3表达水平与细胞自噬水平显著相关:低表达AQP3后,MGC803-AQP3low及SGC7901-AQP3low自噬水平减弱;高表达 AQP3 后,AGS-AQP3high自噬水平增强。3.联合CQ处理后,胃癌细胞对cDDP的敏感性增强。CQ可以逆转AQP3诱导的胃癌细胞对cDDP耐药。结论AQP3通过促进胃癌细胞自噬增强其对顺铂的耐药性,AQP3有望成为胃癌化疗耐药研究的新靶标。
[Abstract]:Background gastric cancer (GastricCancer,GC) is one of the most common malignant tumors in China. Its incidence rate is the highest in digestive tract tumors, and the mortality rate is 2 / 3 of all tumors. It is one of the diseases that seriously threaten the life and health of the people in our country. At present, the treatment of gastric cancer mainly includes radical surgery, chemotherapeutic therapy and molecular targeted therapy. In China, the majority of patients with gastric cancer are advanced when diagnosed, and advanced gastric cancer patients must rely on neoadjuvant chemotherapy or postoperative adjuvant chemotherapy to improve their long-term survival, and the emergence of chemotherapeutic resistance often leads to the failure of chemotherapy. The prognosis is seriously affected. Cisplatin (cisplatin,cDDP) is one of the first-line chemotherapeutic agents for gastric cancer, and its drug resistance is on the rise. Therefore, it is one of the important strategies to improve the curative effect of gastric cancer to study the drug resistance mechanism of gastric cancer to cisplatin chemotherapy and to reduce the resistance of gastric cancer to cisplatin. Aquaporin 3 (aquaporin3,AQP3) is one of the members of aquaporin family (aquaporins,AQPs), which can mediate osmotic pressure dependent transmembrane water transport, and also has permeability to glycerol and other small molecules. Our previous studies have shown that overexpression of AQP3 in gastric cancer may play an important role in carcinogenesis and progression of gastric cancer. The up-regulation of AQP3 may promote GC by promoting epithelial-mesenchymal transformation of (EMT) and stem cell dryness of gastric cancer cells. Invasion and metastasis of cells. However, the role of AQP3 in chemotherapeutic resistance of gastric cancer remains unclear. Autophagy (autophagy) is a process in which macromolecules and organelles in the cytoplasm of eukaryotic cells are transported to lysosomes for isolation and degradation, thus realizing the metabolic needs of the cells themselves and the renewal of some organelles. Autophagy is a common phenomenon in physiological and pathological conditions. Autophagy is not only involved in maintaining cell homeostasis, but also closely related to tumor development and drug resistance. A deeper understanding of the role of AQP3 in promoting autophagy of gastric cancer cells is helpful to elucidate the mechanism of chemoresistance of gastric cancer, to further clarify the role of AQP3 in chemotherapeutic resistance of gastric cancer, and to provide theoretical and experimental basis for AQP3 as a target of gastric cancer therapy. Method 1. Three gastric cancer cell lines, MGC803,SGC7901 and AGS, were selected and transfected with lentivirus, respectively. MGC803 and SGC7901 were transfected with low expression of AQP3 (labeled MGC803-AQP3low,SGC7901-AQP3low) and high expression of AQP3 (labeled AGS-AQP3high). 2. CCK-8 cytotoxicity test was used to detect the sensitivity of cells with different levels of AQP3 expression to cDDP and to the combination of cDDP and chloroquine (chloroquine,CQ). The expression of AQP3 and autophagy related protein was detected by Western blot, laser confocal method. Result 1. The expression of AQP3 in gastric cancer cells was significantly correlated with its sensitivity to cisplatin: the sensitivity of MGC803-AQP3low and SGC7901-AQ3low to cDDP was increased after low expression of AQP3, and the sensitivity of AGS-AQP3high to cDDP was decreased after high expression of AQP3. There was a significant correlation between the expression of AQP3 and autophagy in gastric cancer cells: the decrease of MGC803-AQP3low and SGC7901-AQP3low autophagy after low expression of AQP3, and the enhancement of high autophagy level of AGS-AQP3 after high expression of AQP3. After treated with CQ, the sensitivity of gastric cancer cells to cDDP was enhanced. CQ could reverse the drug resistance of gastric cancer cells induced by AQP3 to cDDP. Conclusion AQP3 can enhance cisplatin resistance by promoting autophagy of gastric cancer cells. AQP3 may be a new target for the study of chemotherapeutic resistance of gastric cancer.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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