P53介导RAD21的亚细胞定位调控自噬促进胃癌化疗抵抗

发布时间：2018-02-03 08:42

本文关键词： RAD21 P53 DNA损伤修复自噬亚细胞定位奥沙利铂敏感性　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:胃癌已成为全球癌症死亡的第二大原因。在中国,胃癌在恶性疾病中排第三。由于症状模糊,多数胃癌患者在确诊时已处于晚期。对于晚期胃癌患者,化疗仍是最重要的治疗方法。铂类,尤其是第三代铂类(奥沙利铂等)已被广泛应用于胃癌临床的一线化疗。然而,由于肿瘤固有或获得性的铂类耐药机制的存在,使得铂类药物的临床疗效不佳。此外,自噬导致的DNA损伤修复是最重要的耐药机制之一。RAD21编码的蛋白是DNA双链断裂修复和核性蛋白质。研究表明,RAD21与化疗药物的敏感性密切相关;RAD21可能成为一个新型的靶点,潜在地通过调节DNA损伤修复增强化疗药物的抗肿瘤效果。自噬参与了化疗药物介导的DNA损伤修复,并且自噬在促进肿瘤的化疗抵抗中发挥了重要的作用。这些证据提示RAD21与自噬可能有着密切的关系。尽管大量研究已证实RAD21在许多肿瘤中表达上调并与化疗药物的敏感性密切相关,但在胃癌中,它与奥沙利铂的药物敏感性关系仍然没有相关报道。本研究中,我们旨在探讨胃癌中RAD21与奥沙利铂药物敏感性的关系,并阐述其具体的发生机制。材料与方法:1.运用PCR、WB检测RAD21在胃癌细胞株和胃癌组织中的表达情况。2.运用MTT、流式及平板克隆检测siRAD21与细胞增殖、凋亡及奥沙利铂的敏感性关系。3.选择47名胃癌患者(对奥沙利铂敏感组)和9名胃癌患者(对奥沙利铂不敏感组),用免疫组化分析胃癌患者RAD21浆表达或核浆表达的无进展生存。4.构建NLS-GV141-RAD21以及NES-GV141-RAD21载体,运用免疫荧光检测RAD21不同的亚细胞定位与奥沙利铂敏感性的关系。5.运用免疫共沉淀及彗星实验探究P53、RAD21及自噬相关的化疗药物敏感性之间的关系。结果:1.各种胃癌细胞株及胃癌组织标本与对照组相比,RAD21表达水平明显上调(p0.05)。2.干扰RAD21可增加胃癌细胞对奥沙利铂的敏感性。3.RAD21的核转位现象与胃癌患者的复发率、临床分期及不良预后密切相关(p0.05)。4.RAD21的核转入载体组与核转出载体组相比,可明显降低胃癌细胞对奥沙利铂的敏感性。5.P53与RAD21呈现共定位并介导了 RAD21的亚细胞定位。6.干扰RAD21联合自噬抑制剂可抑制细胞增殖并最大程度地促使DNA损伤现象的发生。在此过程中,ATG5与RAD21呈明显的正相关(r=0.95,P0.01;r=0.96,P0.01)。结论:RAD21与奥沙利铂的敏感性密切相关;深入机制探讨,P53调控了 RAD21的亚细胞定位,并通过自噬降低了奥沙利铂的敏感性,从而促进了胃癌的进展。综上,这些结果提供了提高胃癌患者对奥沙利铂敏感性的相关策略,使RAD21成为令人瞩目的分子靶点。
[Abstract]:Background & objective: gastric cancer has become the second leading cause of cancer death in the world. Gastric cancer ranks third among malignant diseases in China. Most patients with gastric cancer are in advanced stage at the time of diagnosis. Chemotherapy is still the most important treatment for advanced gastric cancer patients. Especially the third generation platinum (oxaliplatin et al) has been widely used in first-line chemotherapy of gastric cancer. However due to the presence of intrinsic or acquired platinum resistance mechanisms. Make the clinical efficacy of platinum drugs poor. In addition. Repair of DNA damage induced by autophagy is one of the most important mechanisms of drug resistance. The protein encoded by RAD21 is DNA double strand break repair and nuclear protein. RAD21 was closely related to the sensitivity of chemotherapeutic drugs. RAD21 may become a novel target, potentially enhancing the anti-tumor effect of chemotherapeutic drugs by regulating DNA damage repair. Autophagy is involved in chemotherapy-mediated DNA damage repair. Autophagy also plays an important role in promoting chemotherapeutic resistance in tumors. These evidences suggest that RAD21 may be closely related to autophagy. Although numerous studies have confirmed the expression of RAD21 in many tumors. It was up-regulated and closely related to the sensitivity of chemotherapeutic agents. However, there is no correlation between oxaliplatin and oxaliplatin in gastric cancer. In this study, we aim to explore the relationship between RAD21 and oxaliplatin susceptibility in gastric cancer. Materials and methods: 1. The expression of RAD21 in gastric cancer cell line and gastric cancer tissue was detected by PCRX WB. 2. MTT was used. SiRAD21 and cell proliferation were detected by flow cytometry and plate cloning. Apoptosis and oxaliplatin sensitivity. 3. 47 gastric cancer patients (oxaliplatin sensitive group) and 9 gastric cancer patients (oxaliplatin insensitive group) were selected. The expression of RAD21 or nuclear cytoplasm in gastric cancer patients was analyzed by immunohistochemistry. 4. NLS-GV141-RAD21 and NES-GV141-RAD21 vectors were constructed. Using immunofluorescence to detect the relationship between subcellular localization of RAD21 and oxaliplatin sensitivity. 5. To explore p53 by immunoprecipitation and comet assay. The relationship between RAD21 and chemosensitivity of autophagy. Results: 1. All kinds of gastric cancer cell lines and gastric cancer tissue specimens were compared with the control group. The expression level of RAD21 was significantly up-regulated by p0.05. 2. Interfering with RAD21 could increase the sensitivity of gastric cancer cells to oxaliplatin. 3. Nuclear translocation of RAD21 and recurrence rate of gastric cancer patients. Clinical staging and poor prognosis were closely related to p0.05. 4. The nuclear transfer vector group was compared with the nuclear transfer vector group. It can significantly reduce the sensitivity of gastric cancer cells to oxaliplatin. 5.P53 is colocated with RAD21 and mediates the expression of oxaliplatin. The subcellular localization of RAD21. 6. Interfering with RAD21 combined with autophagy inhibitor can inhibit cell proliferation and promote DNA damage to the maximum extent. There was a significant positive correlation between ATG5 and RAD21. Conclusion: the sensitivity of oxaliplatin is closely related to the percentage of 1: RAD21, and the sensitivity of oxaliplatin to oxaliplatin is closely related to the sensitivity of oxaliplatin. To explore the mechanism that P53 regulates the subcellular localization of RAD21 and reduces the sensitivity of oxaliplatin through autophagy, thus promoting the progression of gastric cancer. These results provide strategies to improve the sensitivity of patients with gastric cancer to oxaliplatin and make RAD21 an attractive molecular target.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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