本文選題：前列腺癌　切入點：ELL2　出處：《蘇州大學》2016年博士論文　論文類型：學位論文

【摘要】：背景和目的前列腺癌的發(fā)病率逐年升高,內(nèi)分泌治療在初期能獲得滿意療效,但在一段時間后雄激素阻斷治療變?yōu)闊o效,腫瘤重新開始進展,最終發(fā)展為去勢抵抗性前列腺癌(CRPC),造成腫瘤局部復發(fā),并多伴有全身轉移。一旦當CRPC形成,的目前治療手段療效有限,腫瘤出現(xiàn)會快速轉移,預后極差。因此,闡明前列腺癌發(fā)生進展的分子機制,為前列腺癌的治療提供新的思路和方法有著迫切的臨床需要。ELL2是前列腺癌的一個抑癌基因,在前列腺良性組織內(nèi)高表達,ELL2的缺失或突變可誘導前列腺癌發(fā)生,是前列腺內(nèi)環(huán)境穩(wěn)定的重要因子。而腫瘤的發(fā)生,及其侵襲、轉移、耐藥等生物學行為均與DNA損傷和修復密切相關。本研究旨在明確ELL2對前列腺癌細胞DNA雙鏈損傷的保護作用及探索其對DNA損傷修復的相關調(diào)節(jié)機制,從而為前列腺癌的基礎研究提供新的視點,也為臨床治療前列腺癌提供新的思路。方法1.選取前列腺癌C4-2、LNCa P、PC-3和22RV1細胞株,設計針對ELL2的si RNA,將其和對照si RNA轉染入細胞或者將已構建的Flag-ELL2質(zhì)粒轉染入細胞,再通過不同濃度的阿霉素和γ射線誘導DNA雙鏈損傷,收集細胞行western blot檢測DNA損傷標記物γH2AX的表達,并行彗星實驗檢測彗星的尾距。2.利用基于I-Sce I的DR-GFP同源重組檢測系統(tǒng)和H1299d A3-1#1非同源末端連接檢測系統(tǒng),通過si RNA下調(diào)ELL2表達后,再轉染外源性I-Sce I質(zhì)粒,流式細胞儀檢測GFP陽性細胞百分比,分析ELL2對兩個修復通路的影響。3.將GFP標記的ELL2或GFP,和RFP標記的Ku70、Ku80或RFP轉染入C4-2細胞中,利用Olympus共聚焦激光系統(tǒng)800μW能量的405nm微激光在細胞核原位制DNA雙鏈斷裂,并觀察GFP-ELL2、RFP-Ku70、RFP-Ku80在DNA斷裂末端聚集產(chǎn)生foci的應答效應。4.將Flag-ELL2、GFP-Ku70、GFP-Ku80轉染入HEK293細胞中,進行外源性蛋白質(zhì)免疫共沉淀;將阿霉素處理過和未處理過的C4-2細胞進行核質(zhì)分離提取核蛋白,進行內(nèi)源性蛋白免疫共沉淀,鑒定ELL2是否和Ku70/80蛋白相結合。5.通過si RNA下調(diào)ELL2表達后,將GFP-Ku70或GFP-Ku80轉染入C4-2細胞中,利用微激光制造DNA雙鏈斷裂,在共聚焦顯微鏡下連續(xù)動態(tài)觀察并記錄DNA損傷后8分鐘內(nèi)GFP-Ku70或GFP-Ku80應答形成foci的情況。利用軟件分析foci強度并繪制強度時間曲線。觀察ELL2對Ku70/Ku80在DNA斷端聚集的影響。6.設計只針對內(nèi)源性ELL2而對外源性ELL2沒有作用的si RNA,轉入細胞后,將Flag-ELL2或Flag,和GFP-Ku70/Ku80共同轉入細胞,連續(xù)動態(tài)觀察并記錄微激光制造損傷后8分鐘內(nèi)GFP-Ku70或GFP-Ku80應答形成foci的情況,觀察挽救性上調(diào)ELL2表達后對Ku70/Ku80在DNA斷端聚集的影響。結果1.在前列腺癌多個細胞系中,ELL2表達下調(diào)后,western blot檢測到阿霉素或γ射線誘導的DNA損傷標記物γH2AX表達增加,彗星實驗中慧星尾距明顯增加。ELL2過表達后,γH2AX表達顯著減弱。2.在HR檢測系統(tǒng)中,轉染I-Sce I質(zhì)粒后引起了DNA雙鏈斷裂,并發(fā)生了HR修復,而下調(diào)ELL2表達并不影響DNA雙鏈斷裂的HR修復。在NHEJ檢測系統(tǒng)中,轉染I-Sce I質(zhì)粒后引起了DNA雙鏈斷裂,并發(fā)生了NHEJ修復,下調(diào)ELL2表達使得DNA雙鏈斷裂的NHEJ修復水平減弱。3.ELL2、Ku70、Ku80對微激光誘導的DNA雙鏈斷裂有迅速應答效應,并且ELL2與Ku70/80共同聚集在DNA雙鏈斷裂部位。4.外源性Co-IP顯示ELL2與Ku70、Ku80結合形成蛋白復合物,且ELL2與Ku80的結合程度較Ku70高。內(nèi)源性免疫共沉淀結果顯示阿霉素誘導的DNA損傷促進了ELL2與Ku70、Ku80的結合。5.下調(diào)ELL2的表達使Ku70/80在DNA雙鏈斷裂的斷端聚集形成foci的強度減弱,挽救性過表達ELL2可增強Ku70/80在DNA雙鏈損傷斷端的聚集。結論1.ELL2對前列腺癌細胞的DNA損傷具有修復作用。2.下調(diào)ELL2表達后使DNA雙鏈斷裂的NHEJ修復途徑減弱,而對HR修復途徑?jīng)]有影響。3.ELL2可以和Ku70、Ku80結合形成蛋白復合物,DNA損傷促進了它們的結合。ELL2對Ku70/80蛋白表達水平?jīng)]有影響。4.ELL2和Ku70、Ku80在DNA雙鏈斷裂發(fā)生后,迅速聚集在DNA雙鏈斷裂部位形成foci,并且ELL2可以調(diào)節(jié)Ku70和Ku80在DNA斷端的聚集來調(diào)控NHEJ修復。
[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.

【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.25

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相關期刊論文 前1條

1 Wanqing Chen;Rongshou Zheng;Hongmei Zeng;Siwei Zhang;;The updated incidences and mortalities of major cancers in China, 2011[J];Chinese Journal of Cancer;2015年11期

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本文編號：1646931