MicroRNA-582在膀胱癌中表达的生物学功能及调控机制
发布时间：2018-09-18 11:52
【摘要】：背景与目的膀胱癌是目前泌尿系统常见的恶性肿瘤之一,也是我国发病率最高的泌尿系统肿瘤,且呈逐年增高趋势。膀胱癌的发病原因较复杂,主要有遗传和环境两大因素。吸烟和长期接触化工药品是目前两大环境致病危险因素。其中,吸烟是最为肯定的危险致病因素。研究表明,吸烟可以使膀胱癌发病率增加2-4倍,且危险系数与吸烟强度及吸烟时间成正比。目前,膀胱癌根据浸润深度的不同,临床上多采用不同的治疗方案。浅表性膀胱癌多采用经尿道膀胱肿瘤电切术联合术后膀胱灌注治疗,但术后复发率较高,约60-70%,且复发后有恶性程度增加的趋势。而对于肌层浸润性膀胱癌,临床上目前的标准治疗方法是根治性全膀胱切除术。但是,此手术创伤较大,术后多并发症,且极大的影响患者生活治疗。因此,泌尿外科医生希望明确膀胱癌发生、发展的分子生物学过程,并以此找到能够有效早期诊断、治疗膀胱癌的新方法。MicroRNA是一类小分子非蛋白编码RNA(长度约22个碱基)。它们虽然不能直接编码合成蛋白,但可以通过与靶基因3'UTR部分或完全配对结合抑制其翻译,或造成其特异性降解,以此起到调控基因转录后表达的作用。我们根据前期基因芯片研究发现,miR-582在膀胱尿路上皮癌细胞中呈低表达,通过体外细胞实验,探索miR-582在人膀胱癌细胞中的作用,为膀胱癌的诊断和治疗提供新的靶点。材料、方法及结果第一章miR-582在人膀胱尿路上皮癌中的表达差异方法:1.收集根治性全膀胱切除术后,经病理确认为尿路上皮癌的新鲜肿瘤组织标本为肿瘤(阳性)组及对应的癌旁正常上皮组织,提取组织RNA。2.利用QPCR技术,检测膀胱癌组织及细胞中,miR-582的表达水平。结果:1.与正常癌旁组织相比,膀胱尿路上皮癌组织中,miR-582的表达水平明显下调。2.膀胱尿路上皮癌的组织及细胞中,miR-582呈现稳定低表达,且其表达量与肿瘤的病理分级、临床分期密切相关。第二章miR-582在膀胱癌致病中生物学功能研究方法:1.利用lipofectation 2000将miR-582 mimic瞬时转染进人膀胱癌24、5637细胞中,造成miR-582表达下调。设立对照组(转染miR-NC组)和实验组(转染 miR-582 mimic)。2.转染后的细胞,通过QPCR方法评估转染效率;通过划痕实验,评估膀胱癌细胞的迁移能力;通过Transwell实验,评估膀胱癌细胞的侵袭能力;通过流式细胞技术,评估膀胱癌细胞的干性能力;通过Western Blot实验,评估膀胱癌细胞表征细胞侵干性能力的CD44、S0X2蛋白表达情况。结果:1.转染miR-582mimic后,T24、5637细胞中miR-582的表达明显上调,差异存在统计学意义(P0.05)。2.划痕实验中,转染20h后,miR-582 mimic组的伤口愈合时间明显延长,差异存在统计学意义(P0.05);Transwell实验中,转染miR-582mimic组的T24、5637细胞穿膜细胞数远小于miR-NC mimic组处理细胞,差异存在统计学意义(P0.05);通过流式细胞技术,证实miR-582 mimic组的膀胱癌T24、5637细胞,细胞干性明显下降。第三章miR-582靶向负调控FOXG1表达及FOXG1的作用和意义方法:1.利用miRNA靶基因在线预测软件,预测miR-582下游靶基因,并通过荧光酶素报告基因加以证实。2.在膀胱癌T24、5637细胞中共转染miR-582和过表达F0XG1质粒后进行拯救实验,利用Transwell实验检测膀胱癌细胞侵袭能力的变化,Western blot实验检测蛋白表达变化。3.利用si-FOXG1转染处理人膀胱癌T24、5637细胞株,通过Western blot方法评估转染效率;通过Transwell实验,评估膀胱癌细胞的侵袭能力。结果:1.miRNA靶基因在线预测软件与荧光素酶报告实验结果提示:miR-582在F0XG1基因的3'UTR存在miR-582的互补结合位点。2.转染miR-582于膀胱癌细胞后,膀胱癌细胞侵袭能力下降,而随着外源性F0XG1的加入,侵袭能力增强;MMP2、MMP9等蛋白也在miR-582的影响下表达下调,而随着外源性F0XG1的加入而表达增强。3.Transwell实验中,转染si-FOXG1组的T24、5637细胞穿膜细胞数远小于si-NC组处理细胞,差异存在统计学意义(P0.05)。此结果提示,F0XG1表达下调能够有效抑制膀胱癌细胞的侵袭能力。结论和意义miR-582在膀胱癌组织中表达下调,且其表达量与肿瘤的病理分级、临床分期密切相关,病理分级越高、临床分期越晚的病人,表达量越低。miR-582在膀胱癌细胞中,靶向负调控下游靶基因F0XG1,从而实现起抑制膀胱癌细胞的迁移、侵袭、细胞干性的功能,发挥抑癌基因的作用。本项研究,可以为膀胱癌的分子靶向治疗,提供实验依据。
[Abstract]:BACKGROUND & OBJECTIVE Bladder cancer is one of the most common malignant tumors in the urinary system, and it is also the most common one in China. The incidence of bladder cancer is increasing year by year. Smoking is the most definite risk factor. Studies have shown that smoking can increase the incidence of bladder cancer by 2-4 times, and the risk factor is proportional to the intensity of smoking and smoking time. But the recurrence rate is high, about 60-70%, and the malignant degree is increasing after the recurrence. For myometrial invasive bladder cancer, the current standard treatment is radical cystectomy. Thus, urologists hope to clarify the molecular biological processes involved in the development of bladder cancer, and to find new ways to effectively diagnose and treat bladder cancer. We found that the expression of microRNAs in bladder urothelial carcinoma cells was low. Through in vitro cell experiments, we explored the role of microRNAs in human bladder cancer cells. Materials, Methods and Results Chapter 1 Differential expression of microRNA582 in human bladder urothelial carcinoma Methods: 1. Fresh tumor specimens confirmed by pathology after radical cystectomy were collected as tumor (positive) group and corresponding normal epithelial tissue adjacent to the tumor, and RNA was extracted. Results: 1. Compared with normal adjacent tissues, the expression of microRNAs in bladder urothelial carcinoma tissues was significantly down-regulated. 2. In bladder urothelial carcinoma tissues and cells, the expression of microRNAs-582 was stable and low, and the expression level was correlated with the pathological grade and clinical significance of the tumor. Chapter 2: Biological function of microRNAs in the pathogenesis of bladder cancer: 1. Transient transfection of microRNAs into human bladder cancer 24,5637 cells by lipofection 2000 resulted in the down-regulation of microRNAs-582 expression. Control group (transfected with microRNAs-NC group) and experimental group (transfected with microRNAs-582 mimic) were established. Methods To evaluate the transfection efficiency, to evaluate the migration ability of bladder cancer cells by scratch test, to evaluate the invasion ability of bladder cancer cells by Transwell test, to evaluate the dry ability of bladder cancer cells by flow cytometry, and to evaluate the expression of CD44 and S0X2 protein in bladder cancer cells by Western Blot test. Results: 1. After transfection of Mi-582 mic, the expression of Mi-582 was significantly up-regulated in T24,5637 cells, and the difference was statistically significant (P 0.05). 2. In scratch test, the wound healing time of Mi-582 mic group was significantly prolonged after 20 hours of transfection, and the difference was statistically significant (P 0.05); In Transwell experiment, the transfection of Mi-582 mic group had fine membrane penetration. The number of cells treated with microRNAs was much smaller than that of microRNAs (P 0.05), and the difference was statistically significant (P 0.05). Flow cytometry showed that the cell trunkness of T24,5637 cells in microRNAs group was significantly decreased. Chapter 3: The role and significance of microRNAs targeting negative regulation of FOXG1 expression and FOXG1: 1. Mi-582 downstream target gene was confirmed by luciferase reporter gene. 2. Mi-582 and over-expressed F0XG1 plasmid were co-transfected into bladder cancer T24,5637 cells. Transwell assay was used to detect the invasion ability of bladder cancer cells. Western blot assay was used to detect the expression of protein. 3. Human bladder cancer cells were transfected with si-FOXG1. The transfection efficiency of bladder cancer cell line T24,5637 was evaluated by Western blot, and the invasiveness of bladder cancer cell line T24,5637 was evaluated by Transwell assay. Results: 1. MiRNA target gene online prediction software and luciferase report results indicated that there were complementary binding sites of Mi-582 in the 3'UTR of F0XG1 gene. 2. Mi-582 was transfected in bladder. The invasion ability of bladder cancer cells decreased with the addition of exogenous F0XG1, and increased with the addition of exogenous F0XG1. The expression of MMP2, MMP9 and other proteins was down-regulated under the influence of Mi-582, but increased with the addition of exogenous F0XG1. 3. Transwell experiment showed that the number of transmembrane cells of T24,5637 cells transfected with si-FOXG1 was much smaller than that of si-NC group. The results suggest that the down-regulation of F0XG1 expression can effectively inhibit the invasion of bladder cancer cells. Conclusion The down-regulation of microRNA582 expression in bladder cancer tissue is closely related to the pathological grade and clinical stage of the tumor. The higher the pathological grade, the later the clinical stage, the lower the expression of microRNA582. In bladder cancer cells, microRNA-582 targets the downstream target gene F0XG1, thereby inhibiting the migration, invasion and stem cell function of bladder cancer cells and exerting the role of tumor suppressor genes.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14
，
本文编号：2247824
[Abstract]:BACKGROUND & OBJECTIVE Bladder cancer is one of the most common malignant tumors in the urinary system, and it is also the most common one in China. The incidence of bladder cancer is increasing year by year. Smoking is the most definite risk factor. Studies have shown that smoking can increase the incidence of bladder cancer by 2-4 times, and the risk factor is proportional to the intensity of smoking and smoking time. But the recurrence rate is high, about 60-70%, and the malignant degree is increasing after the recurrence. For myometrial invasive bladder cancer, the current standard treatment is radical cystectomy. Thus, urologists hope to clarify the molecular biological processes involved in the development of bladder cancer, and to find new ways to effectively diagnose and treat bladder cancer. We found that the expression of microRNAs in bladder urothelial carcinoma cells was low. Through in vitro cell experiments, we explored the role of microRNAs in human bladder cancer cells. Materials, Methods and Results Chapter 1 Differential expression of microRNA582 in human bladder urothelial carcinoma Methods: 1. Fresh tumor specimens confirmed by pathology after radical cystectomy were collected as tumor (positive) group and corresponding normal epithelial tissue adjacent to the tumor, and RNA was extracted. Results: 1. Compared with normal adjacent tissues, the expression of microRNAs in bladder urothelial carcinoma tissues was significantly down-regulated. 2. In bladder urothelial carcinoma tissues and cells, the expression of microRNAs-582 was stable and low, and the expression level was correlated with the pathological grade and clinical significance of the tumor. Chapter 2: Biological function of microRNAs in the pathogenesis of bladder cancer: 1. Transient transfection of microRNAs into human bladder cancer 24,5637 cells by lipofection 2000 resulted in the down-regulation of microRNAs-582 expression. Control group (transfected with microRNAs-NC group) and experimental group (transfected with microRNAs-582 mimic) were established. Methods To evaluate the transfection efficiency, to evaluate the migration ability of bladder cancer cells by scratch test, to evaluate the invasion ability of bladder cancer cells by Transwell test, to evaluate the dry ability of bladder cancer cells by flow cytometry, and to evaluate the expression of CD44 and S0X2 protein in bladder cancer cells by Western Blot test. Results: 1. After transfection of Mi-582 mic, the expression of Mi-582 was significantly up-regulated in T24,5637 cells, and the difference was statistically significant (P 0.05). 2. In scratch test, the wound healing time of Mi-582 mic group was significantly prolonged after 20 hours of transfection, and the difference was statistically significant (P 0.05); In Transwell experiment, the transfection of Mi-582 mic group had fine membrane penetration. The number of cells treated with microRNAs was much smaller than that of microRNAs (P 0.05), and the difference was statistically significant (P 0.05). Flow cytometry showed that the cell trunkness of T24,5637 cells in microRNAs group was significantly decreased. Chapter 3: The role and significance of microRNAs targeting negative regulation of FOXG1 expression and FOXG1: 1. Mi-582 downstream target gene was confirmed by luciferase reporter gene. 2. Mi-582 and over-expressed F0XG1 plasmid were co-transfected into bladder cancer T24,5637 cells. Transwell assay was used to detect the invasion ability of bladder cancer cells. Western blot assay was used to detect the expression of protein. 3. Human bladder cancer cells were transfected with si-FOXG1. The transfection efficiency of bladder cancer cell line T24,5637 was evaluated by Western blot, and the invasiveness of bladder cancer cell line T24,5637 was evaluated by Transwell assay. Results: 1. MiRNA target gene online prediction software and luciferase report results indicated that there were complementary binding sites of Mi-582 in the 3'UTR of F0XG1 gene. 2. Mi-582 was transfected in bladder. The invasion ability of bladder cancer cells decreased with the addition of exogenous F0XG1, and increased with the addition of exogenous F0XG1. The expression of MMP2, MMP9 and other proteins was down-regulated under the influence of Mi-582, but increased with the addition of exogenous F0XG1. 3. Transwell experiment showed that the number of transmembrane cells of T24,5637 cells transfected with si-FOXG1 was much smaller than that of si-NC group. The results suggest that the down-regulation of F0XG1 expression can effectively inhibit the invasion of bladder cancer cells. Conclusion The down-regulation of microRNA582 expression in bladder cancer tissue is closely related to the pathological grade and clinical stage of the tumor. The higher the pathological grade, the later the clinical stage, the lower the expression of microRNA582. In bladder cancer cells, microRNA-582 targets the downstream target gene F0XG1, thereby inhibiting the migration, invasion and stem cell function of bladder cancer cells and exerting the role of tumor suppressor genes.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14
，
本文编号：2247824
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