自噬标志物ATG5与LC3在肾癌组织中的表达及其对肾癌耐药性的影响研究

发布时间：2018-05-03 18:45

本文选题：自噬 + 肾癌　；参考：《第三军医大学》2017年硕士论文

【摘要】：背景肾透明细胞癌约占成人恶性肿瘤的2%~3%,截止2016年的统计学数据显示肾癌仍是我国发病率约第10位的恶性肿瘤,为成人肾脏恶性肿瘤的主要部分。近些年来其发病率呈逐年上涨趋向,全球每年约有10万人死于肾癌,肾癌分布总体上发病年龄可见于各年龄段,高发年龄为50~70岁,最近流行病学统计数据显示男性肾癌发病率略多于女性,地区差异也比较明显,城市稍高于农村。目前肾癌因缺乏早期特异性的诊断方法导致大部分患者诊断时肿瘤已发展至中晚期。尽管现在对肾癌的认识有了很大的进步,诊疗手段也日益增多,但由于肾癌发生发展机制仍未明确,故肾癌目前仍以手术治疗为主,术后患者生存率仍不太理想。目前,还没有公认的可用于临床诊断肾癌的肿瘤标志物。肾癌的术前诊断主要依靠腹部影像学检查,然而,因影像学诊断的限制,腹部CT一般只能发现直径大于5mm以上的肾癌,并且确诊还需病理学检查。因此,探索肾癌可能发生发展及耐药的分子机制对肾癌的诊断、治疗及预后评估具有重要意义。目前研究已证明,细胞自噬主要有三种存在形式,包括大自噬(macroautophagy,MA)、分子伴侣自噬(chaperone-mediated autophagy,CMA)和小自噬(microautophagy)。大自噬(macroautophagy,MA)即通常所说的自噬,是使用双层脂质膜结构包裹胞内短效周期蛋白形成自噬泡,并且与次级溶酶体(溶酶体)融合后,在溶酶体内酸性蛋白降解酶的作用下进行降解再利用。一般在体外细胞培养中,细胞经过血清饥饿诱导短期内(4-6小时)可以达到高峰。对于小自噬目前研究相对较少,主要是指是通过溶酶体直接与目的蛋白进行融合后降解。近来研究发现,自噬可以通过代谢自身蛋白质和细胞器来逃避外界应激,以此来维持细胞稳态并促进细胞存活,新近发现,肿瘤细胞往往通过自噬来增强其对抗癌药物的耐药性,以此逃避肿瘤药物的杀伤作用。自噬相关基因5(ATG5)作为常用自噬检测关键基因之一,其表达水平与自噬明显相关。微管相干蛋白1轻链3(LC3)是目前研究最多的自噬标志性基因之一,其共同参与自噬体的形成,是自噬特异的常用诊断标志物之一。目前,自噬的检测方法主要包括免疫组织化学中检测自噬体的形成、western blot检测LC3。细胞增殖情况常用的检测技术包括MTT法、流式细胞术等。目前,自噬在肾癌中尤其是其耐药性的研究尚不深入,本研究采用免疫组织化学染色、慢病毒感染、Western Blot、MTT、流式细胞术等多种实验方法和技术,从分子和细胞水平,观察了大自噬在体外肾癌细胞增殖及舒尼替尼耐药性中的作用及其分子机制。主要研究结果:一、ATG5、LC3在肾透明细胞癌组织中高表达,且与肾透明细胞癌的分级及预后相关1.对99例肾癌组织和17例癌旁组织进行免疫组化染色分析发现,与癌旁组织相比,ATG5和LC3在肾透明细胞癌组织中表达量明显升高。临床病理资料统计分析发现,ATG5和LC3的表达高低与年龄、性别、肿瘤部位无统计学差异,但与肾癌肿瘤恶性程度、TNM分期密切相关;随着病理学分级和TNM临床分期的增加,ATG5和LC3的表达依次升高。并且,ATG5高表达患者生存率较低,预后较差。二、大自噬促进A-498肾癌细胞株增殖1.通过慢病毒介导的ATG5 shRNA表达载体感染构建低表达的A-498肾癌细胞株,并使用Western Blot相关技术进行验证,成功构建了ATG5低表达肾癌细胞株;2.流式细胞术检测ATG5正常表达与低表达组中A-498细胞增殖的变化;3.7天MTT增殖曲线结果显示,敲低A-498肾透明细胞癌株中ATG5表达,能显著降低肾癌细胞的增殖能力。4.采用H_2O_2刺激及细胞活力检测证明,在氧应激情况下,敲低A-498肾癌细胞株中ATG5表达,能显著降低肾癌细胞的存活率。三、抑制自噬能降低肾癌细胞对舒尼替尼的耐药性通过MTT实验发现,ATG5敲低的A-498肾癌细胞对舒尼替尼药物的耐药性增加。本文结论:自噬可通过抑制ATG5介导的自噬信号通路来刺激肾癌细胞增殖,本课题为肾癌增殖及治疗中耐药性的研究提供了新的思路,并为临床肾癌的诊治提供新的可能靶点和线索。
[Abstract]:Background renal clear cell carcinoma accounts for about 2%~3% of adult malignant tumors. Statistical data in 2016 show that renal cancer is still the main part of the malignant tumor in China, which is the main part of the adult malignant tumor. In recent years, the incidence of renal cancer is increasing year by year, about 100 thousand people die from renal cancer in the world and the distribution of renal cancer is on the whole. The age of onset is seen in all ages. The age of high incidence is 50~70 years. Recent epidemiological statistics show that the incidence of male kidney cancer is slightly more than that of women. There has been a great progress in the understanding of renal cancer, and the means of diagnosis and treatment are increasing. However, the mechanism of renal cancer is still not clear, so the renal carcinoma is still mainly operated on. The survival rate of the patients is still not ideal. At present, there is no recognized tumor marker for clinical diagnosis of renal cancer. The preoperative diagnosis of renal cancer depends mainly on the diagnosis of renal cancer. Abdominal imaging examination, however, because of the limitation of imaging diagnosis, abdominal CT can only find more than 5mm more than 5mm of renal cancer, and the diagnosis is still necessary for pathological examination. Therefore, it is of great significance to explore the molecular mechanism of the possible development and resistance of renal cancer to the diagnosis, treatment and prognosis of renal cancer. There are three main forms of existence, including macroautophagy (MA), chaperone-mediated autophagy (CMA), and small autophagy (microautophagy). The autophagy (macroautophagy, MA) is usually described as autophagy, which uses a double lipid membrane structure to encapsulate short acting cyclin and form a autophagic bubble, and it is associated with a secondary lysase. After fusion, the body (Rong Meiti) is degraded and reused under the action of the enzyme in vivo acidic protein degrading enzyme. In vitro culture, the cells are induced by serum starvation in the short term (4-6 hours) to reach the peak. The present study is relatively small for the small autophagy, mainly referring to the direct fusion of the target protein through Rong Meiti. Late degradation. Recent studies have found that autophagy can avoid external stress by metabolism of its own proteins and organelles to maintain cell homeostasis and promote cell survival. Recently, tumor cells tend to enhance their resistance to anticancer drugs by autophagy to escape the killing effect of tumor drugs. Autophagy related gene 5 (ATG5) As one of the key genes of autophagy detection, the expression level is obviously related to autophagy. Microtubule coherent protein 1 light chain 3 (LC3) is one of the most widely studied autophagic marker genes. It participates in the formation of autophagic and is one of the most commonly used diagnostic markers for autophagy. In the study, the formation of autophago is detected. The common detection techniques for the detection of LC3. cell proliferation by Western blot include MTT and flow cytometry. At present, the study of autophagy in renal carcinoma, especially its drug resistance, is not deep. This study uses immunohistochemical staining, lentivirus infection, Western Blot, MTT, flow cytometry and other experimental parties. Methods and techniques, from molecular and cellular levels, observed the role of autophagy in the proliferation of renal cell carcinoma cells in vitro and its molecular mechanism. The main results are: 1, ATG5, LC3 are highly expressed in the tissues of renal clear cell carcinoma, and are related to the classification and prognosis of renal clear cell carcinoma in 1. pairs of renal carcinoma tissues and 17 para cancerous tissues Immunohistochemical staining showed that the expression of ATG5 and LC3 in the tissues of renal clear cell carcinoma was significantly higher than that of the para cancerous tissue. The statistical analysis of clinicopathological data found that the expression of ATG5 and LC3 had no statistical difference with age, sex and tumor location, but it was closely related to the malignant degree of renal cancer and TNM staging. The expression of ATG5 and LC3 increased in sequence and TNM, and the survival rate of the ATG5 high expression patients was low and the prognosis was poor. Two, the large autophagy promoted the proliferation of A-498 renal cancer cell line 1. through the lentivirus mediated ATG5 shRNA expression vector to construct the low expression A-498 renal cancer cell line, and used Western Blot related techniques. The ATG5 low expression renal cell carcinoma cell line was successfully constructed, and the 2. flow cytometry was used to detect the changes in the proliferation of A-498 cells in normal ATG5 expression and low expression group. The 3.7 day MTT proliferation curve showed that the expression of ATG5 in low A-498 renal cell carcinoma cells could significantly reduce the proliferation ability of renal cell carcinoma cell.4. by H_2O_2 stimulation and cell viability detection. It was proved that ATG5 expression in low A-498 renal cell carcinoma cells could significantly reduce the survival rate of renal cell carcinoma cells under oxygen stress. Three, inhibition of autophagy could reduce the resistance of renal cancer cells to suneinib by MTT test. The drug resistance of ATG5 cells with low ATG5 knockdown was increased. This conclusion: autophagy can be inhibited by inhibition. ATG5 mediated autophagy signaling pathway to stimulate the proliferation of renal cell carcinoma cells. This topic provides new ideas for the study of the proliferation and drug resistance of renal cancer and provides new possible targets and clues for the diagnosis and treatment of renal cancer.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11

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