ELL2在前列腺癌细胞DNA损伤修复中的作用及其机制的实验研究

发布时间：2018-03-22 03:38

本文选题：前列腺癌　切入点：ELL2　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：背景和目的前列腺癌的发病率逐年升高,内分泌治疗在初期能获得满意疗效,但在一段时间后雄激素阻断治疗变为无效,肿瘤重新开始进展,最终发展为去势抵抗性前列腺癌(CRPC),造成肿瘤局部复发,并多伴有全身转移。一旦当CRPC形成,的目前治疗手段疗效有限,肿瘤出现会快速转移,预后极差。因此,阐明前列腺癌发生进展的分子机制,为前列腺癌的治疗提供新的思路和方法有着迫切的临床需要。ELL2是前列腺癌的一个抑癌基因,在前列腺良性组织内高表达,ELL2的缺失或突变可诱导前列腺癌发生,是前列腺内环境稳定的重要因子。而肿瘤的发生,及其侵袭、转移、耐药等生物学行为均与DNA损伤和修复密切相关。本研究旨在明确ELL2对前列腺癌细胞DNA双链损伤的保护作用及探索其对DNA损伤修复的相关调节机制,从而为前列腺癌的基础研究提供新的视点,也为临床治疗前列腺癌提供新的思路。方法1.选取前列腺癌C4-2、LNCa P、PC-3和22RV1细胞株,设计针对ELL2的si RNA,将其和对照si RNA转染入细胞或者将已构建的Flag-ELL2质粒转染入细胞,再通过不同浓度的阿霉素和γ射线诱导DNA双链损伤,收集细胞行western blot检测DNA损伤标记物γH2AX的表达,并行彗星实验检测彗星的尾距。2.利用基于I-Sce I的DR-GFP同源重组检测系统和H1299d A3-1#1非同源末端连接检测系统,通过si RNA下调ELL2表达后,再转染外源性I-Sce I质粒,流式细胞仪检测GFP阳性细胞百分比,分析ELL2对两个修复通路的影响。3.将GFP标记的ELL2或GFP,和RFP标记的Ku70、Ku80或RFP转染入C4-2细胞中,利用Olympus共聚焦激光系统800μW能量的405nm微激光在细胞核原位制DNA双链断裂,并观察GFP-ELL2、RFP-Ku70、RFP-Ku80在DNA断裂末端聚集产生foci的应答效应。4.将Flag-ELL2、GFP-Ku70、GFP-Ku80转染入HEK293细胞中,进行外源性蛋白质免疫共沉淀;将阿霉素处理过和未处理过的C4-2细胞进行核质分离提取核蛋白,进行内源性蛋白免疫共沉淀,鉴定ELL2是否和Ku70/80蛋白相结合。5.通过si RNA下调ELL2表达后,将GFP-Ku70或GFP-Ku80转染入C4-2细胞中,利用微激光制造DNA双链断裂,在共聚焦显微镜下连续动态观察并记录DNA损伤后8分钟内GFP-Ku70或GFP-Ku80应答形成foci的情况。利用软件分析foci强度并绘制强度时间曲线。观察ELL2对Ku70/Ku80在DNA断端聚集的影响。6.设计只针对内源性ELL2而对外源性ELL2没有作用的si RNA,转入细胞后,将Flag-ELL2或Flag,和GFP-Ku70/Ku80共同转入细胞,连续动态观察并记录微激光制造损伤后8分钟内GFP-Ku70或GFP-Ku80应答形成foci的情况,观察挽救性上调ELL2表达后对Ku70/Ku80在DNA断端聚集的影响。结果1.在前列腺癌多个细胞系中,ELL2表达下调后,western blot检测到阿霉素或γ射线诱导的DNA损伤标记物γH2AX表达增加,彗星实验中慧星尾距明显增加。ELL2过表达后,γH2AX表达显著减弱。2.在HR检测系统中,转染I-Sce I质粒后引起了DNA双链断裂,并发生了HR修复,而下调ELL2表达并不影响DNA双链断裂的HR修复。在NHEJ检测系统中,转染I-Sce I质粒后引起了DNA双链断裂,并发生了NHEJ修复,下调ELL2表达使得DNA双链断裂的NHEJ修复水平减弱。3.ELL2、Ku70、Ku80对微激光诱导的DNA双链断裂有迅速应答效应,并且ELL2与Ku70/80共同聚集在DNA双链断裂部位。4.外源性Co-IP显示ELL2与Ku70、Ku80结合形成蛋白复合物,且ELL2与Ku80的结合程度较Ku70高。内源性免疫共沉淀结果显示阿霉素诱导的DNA损伤促进了ELL2与Ku70、Ku80的结合。5.下调ELL2的表达使Ku70/80在DNA双链断裂的断端聚集形成foci的强度减弱,挽救性过表达ELL2可增强Ku70/80在DNA双链损伤断端的聚集。结论1.ELL2对前列腺癌细胞的DNA损伤具有修复作用。2.下调ELL2表达后使DNA双链断裂的NHEJ修复途径减弱,而对HR修复途径没有影响。3.ELL2可以和Ku70、Ku80结合形成蛋白复合物,DNA损伤促进了它们的结合。ELL2对Ku70/80蛋白表达水平没有影响。4.ELL2和Ku70、Ku80在DNA双链断裂发生后,迅速聚集在DNA双链断裂部位形成foci,并且ELL2可以调节Ku70和Ku80在DNA断端的聚集来调控NHEJ修复。
[Abstract]:Background and objective: the incidence of prostate cancer increased year by year, endocrine therapy can obtain satisfactory curative effect at the early stage, but in a period of time after androgen deprivation therapy becomes invalid, tumor start progress, ultimately the development of castration resistant prostate cancer (CRPC), resulting in local tumor recurrence and metastasis, accompanied by systemic. Once the formation of CRPC at present, the treatment effect is limited, there will be rapid tumor metastasis, poor prognosis. Therefore, elucidating the molecular mechanism underlying the progression of prostate cancer, for the treatment of prostate cancer to provide new ideas and methods are in urgent need of clinical.ELL2 is a tumor suppressor gene for prostate cancer, high expression in benign prostate tissues. The loss or mutation of ELL2 can induce prostate cancer, is an important factor for stable environment within the prostate. And the occurrence of tumor invasion and metastasis, and the biological behavior of drug resistance and DNA Damage and repair are closely related. The purpose of this study is to explore the localization of ELL2 and DNA damage repair related regulation mechanism of protective effect of DNA prostate cancer cells double strand damage, so as to provide a new perspective for the basic research of prostate cancer, but also provide new ideas for clinical treatment of prostate cancer. Methods 1. prostate cancer C4-2. LNCa P, PC-3 and 22RV1 cell lines, Si RNA designed for ELL2, and the control Si RNA was transfected into the cells or the constructed Flag-ELL2 plasmid was transfected into the cells by different concentrations of adriamycin and gamma ray induced DNA double strand damage, the expression of Western blot cells were collected for detection of DNA damage markers gamma H2AX the comet assay, parallel comet tail from.2. using I-Sce I DR-GFP H1299d A3-1#1 homologous recombination detection system and non homologous end joining detection system based on Si RNA by down regulating the expression of ELL2, then With exogenous I-Sce I plasmid and detected the percentage of GFP positive cells by flow cytometry and the analysis of effect of ELL2 on the two repair pathway of.3. GFP labeled ELL2 or GFP, and RFP markers Ku70, Ku80 or RFP were transfected into C4-2 cells, 405nm 800 W energy Olympus using confocal laser micro laser system in the nucleus in situ synthesis of DNA double strand breaks, and observe the GFP-ELL2, RFP-Ku70,.4. RFP-Ku80 DNA in response to fault end aggregated into foci Flag-ELL2, GFP-Ku70, GFP-Ku80 were transfected into HEK293 cells, exogenous protein immunoprecipitation; the adriamycin treated and untreated C4-2 cells were isolated from the nucleoplasm nuclear protein, endogenous CO immunoprecipitation, determine whether ELL2 and Ku70/80 protein combined with.5. by Si RNA can downregulate ELL2 expression, GFP-Ku70 or GFP-Ku80 were transfected into C4-2 cells, the use of micro laser manufacturing DNA double chain Fracture in the confocal microscope and the dynamic observation of foci GFP-Ku70 within 8 minutes or GFP-Ku80 response form after DNA injury record. Foci analysis software and drawing strength using time intensity curve. The effect of ELL2 on Ku70/Ku80 in DNA end.6. aggregation design only for the endogenous ELL2 and exogenous ELL2 without si the role of RNA, into cells, Flag-ELL2 or Flag, and GFP-Ku70/Ku80 into cells, continuous and dynamic observation and recording of micro laser damage within 8 minutes after GFP-Ku70 or GFP-Ku80 response to form foci, observe the rescue after the up-regulated expression of ELL2 on Ku70/Ku80 in the stump of DNA aggregation. Results 1. in multiple cells Department of prostate cancer, the down-regulation of ELL2, Western blot to detect DNA damage markers induced by Adriamycin or gamma gamma H2AX expression increased, the comet assay in comet tail distance increased significantly.ELL2 After the expression of gamma H2AX expression decreased in.2. HR detection system, caused DNA double strand breaks after transfection of I-Sce I plasmid, and HR repair, and the expression of ELL2 did not affect HR repair of DNA double strand breaks in NHEJ detection system, caused DNA double strand breaks after transfection of I-Sce I plasmid and, NHEJ repair, down-regulation of ELL2 NHEJ expression level makes the repair of DNA double strand breaks of Ku70, reduced.3.ELL2, Ku80 has a rapid response effect on micro laser induced DNA double strand breaks, and ELL2 and Ku70/80 together in DNA double strand break sites.4. exogenous Co-IP display ELL2 and Ku70, Ku80 combined with the formation of protein complexes, and binding of ELL2 with Ku80 was higher than that of Ku70. The results showed that DNA damage induced by adriamycin promoted ELL2 and Ku70 endogenous CO immunoprecipitation combined with down-regulation of.5. expression ELL2 Ku80 Ku70/80 in the broken ends of DNA double strand breaks in the formation of poly foci The intensity, save the over expression of ELL2 can enhance the Ku70/80 broken end gathered in DNA double strand damage. Conclusion 1.ELL2 DNA damage on prostate cancer cells can restore the expression of ELL2.2. after NHEJ DNA double strand break repair pathway decreased, while.3.ELL2 had no effect on HR and Ku70 repair pathways, Ku80 binding the formation of protein complexes, DNA damage promoted their binding to.ELL2 on the expression level of Ku70/80 protein had no effect on.4.ELL2 and Ku70, Ku80 in DNA double strand break occurs, the rapid accumulation of formation of foci double strand breaks in DNA site, and ELL2 can regulate Ku70 and Ku80 DNA in the broken end of the aggregation to regulate NHEJ repair.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

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