本文選題：膠質(zhì)母細胞瘤　切入點：EGFR　出處：《天津醫(yī)科大學》2015年博士論文　論文類型：學位論文

【摘要】：目的:1.構(gòu)建mi R-21抑制劑的慢病毒系統(tǒng)以及PPARA和VHL的熒光素酶報告質(zhì)粒,驗證PPARA和VHL為mi R-21的靶基因并闡述mi R-21通過PPARA和VHL對EGFR信號通路調(diào)控的詳細分子機制;在體內(nèi)實驗和體外實驗中聯(lián)合mi R-21抑制劑和EGFR單克隆抗體--尼妥珠單克隆抗體治療膠質(zhì)瘤,為優(yōu)化膠質(zhì)瘤的基因治療提供新的思路。2.利用生物信息學方法找出可能受lnc RNA HOTAIR調(diào)控的信號通路,通過小分子抑制劑阻斷以及表達載體構(gòu)建等方法闡明LSD1復(fù)合物和PRC2復(fù)合物在lnc RNA HOTAIR對膠質(zhì)瘤細胞周期進展調(diào)控中的分子機制。方法:1.用表達mi R-21抑制劑的慢病毒感染膠質(zhì)瘤細胞系U87、LN229和U251,Western blot方法檢測EGFR、p-EGFR、AKT、p-AKT的表達水平變化;細胞克隆形成實驗、細胞流式分析以及transwell實驗檢測mi R-21對膠質(zhì)瘤細胞增殖、細胞周期進展、細胞侵襲以及細胞凋亡的影響。2.通過靶點預(yù)測找出mi R-21可能的靶點;熒光素酶報告實驗驗證PPARA和VHL為mi R-21的靶基因;免疫熒光和蛋白質(zhì)免疫共沉淀揭示β-catenin和PPARα之間的調(diào)控網(wǎng)絡(luò)。3.構(gòu)建膠質(zhì)瘤顱內(nèi)動物模型,研究mi R-21抑制劑和尼妥珠單抗聯(lián)合應(yīng)用的體內(nèi)抑瘤效果。4.生物信息學分析HOTAIR可能調(diào)控的下游信號傳導(dǎo)通路;利用含lnc RNA HOTAIR干擾序列的慢病毒、EZH2小分子抑制劑DZNep、LSD1小分子抑制劑2-PCPA分別處理膠質(zhì)瘤細胞U87和LN229,分析它們對膠質(zhì)瘤細胞周期的影響。5.在敲低HOTAIR的U87和LN229細胞系中分別過表達HOTAIR的3’及5’結(jié)構(gòu)域,驗證si RNA HOTAIR引起的細胞周期阻滯能否部分恢復(fù)。6.構(gòu)建顱內(nèi)原位膠質(zhì)瘤動物模型,檢測HTOAIR siRNA的體內(nèi)抑瘤效果。結(jié)果:1.感染mi R-21抑制劑病毒的膠質(zhì)瘤細胞中p-EGFR和p-AKT表達明顯下調(diào),細胞的增殖受到抑制,細胞周期被阻滯在G1期,細胞凋亡明顯增加(p0.01)。2.Western blot實驗和熒光素酶報告實驗證實VHL及PPARA為mi R-21的直接作用靶點。3.感染mi R-21抑制劑或轉(zhuǎn)染VHL表達質(zhì)粒均能降低經(jīng)典型Wnt/β-catenin信號通路的活性。Western blot和免疫熒光結(jié)果提示mi R-21通過靶定PPARα進而上調(diào)轉(zhuǎn)錄因子AP-1的轉(zhuǎn)錄活性調(diào)節(jié)EGFR/AKT信號通路。4.蛋白質(zhì)免疫共沉淀結(jié)果證明β-catenin和AP-1能形成復(fù)合體,且復(fù)合體的形成受mi R-21的調(diào)控。5.mi R-21抑制劑聯(lián)合尼妥珠單抗治療膠質(zhì)瘤效果優(yōu)于單藥治療。6.生物信息學結(jié)果顯示HOTAIR調(diào)節(jié)的基因主要參與細胞周期的調(diào)控。7.EZH2抑制劑DZNep能模擬si RNA HOTAIR的功能,將膠質(zhì)瘤細胞周期阻滯在G1期。8.HOTAIR敲低的細胞系中過表達其5’結(jié)構(gòu)域能部分的回復(fù)受si RNA HOTAIR引起的細胞周期抑制作用。9.膠質(zhì)瘤顱內(nèi)模型證實siRNA HOTAIR能抑制膠質(zhì)瘤的生長。結(jié)論:1.mi R-21通過靶定VHL/β-catenin和PPARα/AP-1調(diào)節(jié)EGFR/AKT信號傳導(dǎo)通路。2.EGFR和mi R-21之間存在反饋調(diào)節(jié)環(huán)路。3.體內(nèi)外研究顯示聯(lián)合尼妥珠單克隆抗體和mi R-21抑制劑治療膠質(zhì)瘤優(yōu)于單藥治療。4.Lnc RNA HOTAIR調(diào)控的基因主要參與細胞周期過程;HOTAIR主要通過其5’結(jié)構(gòu)域和PRC2復(fù)合物的結(jié)合調(diào)節(jié)膠質(zhì)瘤細胞周期進展。5.敲低HOTAIR的表達能在體內(nèi)抑制膠質(zhì)瘤的惡性增殖。
[Abstract]:Objective: to construct mi 1. R-21 inhibitors and PPARA lentiviral system and VHL luciferase reporter plasmid, verification of PPARA and VHL as the target gene of MI R-21 and MI R-21 by PPARA and VHL on the regulation of EGFR signaling pathway with molecular mechanism; in vivo and in vitro experiments in MI R-21 inhibitors and EGFR monoclonal antibody nimotuzumab treatment of glioma, and provide new ideas using.2. biological information by LNC signaling pathway may find RNA HOTAIR control method for gene therapy of glioma by optimization of small molecule inhibitors block and expression vector construction method of LSD1 complex and PRC2 complex in LNC RNA HOTAIR on the molecular mechanism of progress glioma cell cycle regulation. Methods: 1. expression by Mi inhibitors of R-21 lentivirus infected glioma cell lines U87, LN229 and U251, Western blot EGFR detection method , p-EGFR, AKT, p-AKT expression level changes; cell clone formation assay, flow cytometry analysis and Transwell assay of MI R-21 on glioma cell proliferation, cell cycle progression, invasion and apoptosis effect of.2. cells to identify targets of MI R-21 may be through the target prediction; luciferase reporter experiments of PPARA and VHL the target gene mi R-21; reveal the regulation between.3. and PPAR network construction of alpha beta -catenin glioma animal model of intracranial immunofluorescence and immunoprecipitation of MI protein, and R-21 inhibitor nimotuzumab combined in vivo antitumor effect of.4. bioinformatics analysis of the downstream HOTAIR signaling pathway may regulate the LNC containing RNA; HOTAIR interference sequence with lentivirus, EZH2 small molecule inhibitor DZNep, small molecule inhibitors of LSD1 were treated with 2-PCPA U87 and LN229 glioma cells, analysis of glioma fine Effect of.5. cell cycle in U87 and LN229 cell lines at low HOTAIR were over expression of HOTAIR 3 'and 5' domain, can verify the Si RNA cell cycle arrest caused by HOTAIR partially restored.6. construction in situ intracranial glioma animal model, detection of HTOAIR siRNA in vivo antitumor effect. Results: the down-regulation of p-EGFR and p-AKT expression in glioma cells infected with MI virus R-21 1. inhibitor, cell proliferation was inhibited, cell cycle arrest in G1 phase, cell apoptosis increased significantly (P0.01).2.Western blot experiment and luciferase reporter assay demonstrated that VHL PPARA and MI R-21 for the direct targets of.3. infection or transfection of VHL R-21 inhibitor mi the expression plasmid could reduce the transcriptional activity of.Western regulating activity of blot and immunofluorescence results of classical Wnt/ beta -catenin signaling pathway mi R-21 by targeting PPAR alpha and upregulation of the transcription factor AP-1 Festival The results prove that -catenin and AP-1 can form complex beta EGFR/AKT signaling pathway.4. protein co immunoprecipitation, and complex formation is better than the control effect of.5.mi R-21 inhibitor combined with nimotuzumab treatment of glioma by Mi R-21 monotherapy.6. bioinformatics results showed that regulation of.7.EZH2 inhibitor DZNep HOTAIR regulating genes mainly involved in cell cycle can simulation of Si RNA HOTAIR, the glioma cell cycle arrest in G1 phase of.8.HOTAIR knockdown cell lines overexpressing the 5 'domain can reply by cell cycle Si RNA HOTAIR induced inhibition of.9. glioma model of intracranial confirmed that siRNA HOTAIR can inhibit the growth of glioma. Conclusion: 1.mi by R-21 target feedback loop.3. in vitro and in vivo studies have shown that combination existed between VHL/ and PPAR alpha /AP-1 beta -catenin regulating EGFR/AKT signal transduction pathways of.2.EGFR and MI R-21 With MI monoclonal antibody and R-21 inhibitor for the treatment of glioma is superior to monotherapy.4.Lnc RNA HOTAIR regulated genes mainly involved in cell cycle process; combined with the expression of HOTAIR mainly through the 5 'domain and PRC2 complexes regulate glioma cell cycle progression.5. knockdown of HOTAIR in vivo inhibition of glioma malignant proliferation.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R739.4

【共引文獻】

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本文編號：1637446