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人胶质瘤中miRNA-21及LncRNA HOTAIR的调控机制研究

发布时间:2018-03-20 04:13

  本文选题:胶质母细胞瘤 切入点:EGFR 出处:《天津医科大学》2015年博士论文 论文类型:学位论文


【摘要】:目的:1.构建mi R-21抑制剂的慢病毒系统以及PPARA和VHL的荧光素酶报告质粒,验证PPARA和VHL为mi R-21的靶基因并阐述mi R-21通过PPARA和VHL对EGFR信号通路调控的详细分子机制;在体内实验和体外实验中联合mi R-21抑制剂和EGFR单克隆抗体--尼妥珠单克隆抗体治疗胶质瘤,为优化胶质瘤的基因治疗提供新的思路。2.利用生物信息学方法找出可能受lnc RNA HOTAIR调控的信号通路,通过小分子抑制剂阻断以及表达载体构建等方法阐明LSD1复合物和PRC2复合物在lnc RNA HOTAIR对胶质瘤细胞周期进展调控中的分子机制。方法:1.用表达mi R-21抑制剂的慢病毒感染胶质瘤细胞系U87、LN229和U251,Western blot方法检测EGFR、p-EGFR、AKT、p-AKT的表达水平变化;细胞克隆形成实验、细胞流式分析以及transwell实验检测mi R-21对胶质瘤细胞增殖、细胞周期进展、细胞侵袭以及细胞凋亡的影响。2.通过靶点预测找出mi R-21可能的靶点;荧光素酶报告实验验证PPARA和VHL为mi R-21的靶基因;免疫荧光和蛋白质免疫共沉淀揭示β-catenin和PPARα之间的调控网络。3.构建胶质瘤颅内动物模型,研究mi R-21抑制剂和尼妥珠单抗联合应用的体内抑瘤效果。4.生物信息学分析HOTAIR可能调控的下游信号传导通路;利用含lnc RNA HOTAIR干扰序列的慢病毒、EZH2小分子抑制剂DZNep、LSD1小分子抑制剂2-PCPA分别处理胶质瘤细胞U87和LN229,分析它们对胶质瘤细胞周期的影响。5.在敲低HOTAIR的U87和LN229细胞系中分别过表达HOTAIR的3’及5’结构域,验证si RNA HOTAIR引起的细胞周期阻滞能否部分恢复。6.构建颅内原位胶质瘤动物模型,检测HTOAIR siRNA的体内抑瘤效果。结果:1.感染mi R-21抑制剂病毒的胶质瘤细胞中p-EGFR和p-AKT表达明显下调,细胞的增殖受到抑制,细胞周期被阻滞在G1期,细胞凋亡明显增加(p0.01)。2.Western blot实验和荧光素酶报告实验证实VHL及PPARA为mi R-21的直接作用靶点。3.感染mi R-21抑制剂或转染VHL表达质粒均能降低经典型Wnt/β-catenin信号通路的活性。Western blot和免疫荧光结果提示mi R-21通过靶定PPARα进而上调转录因子AP-1的转录活性调节EGFR/AKT信号通路。4.蛋白质免疫共沉淀结果证明β-catenin和AP-1能形成复合体,且复合体的形成受mi R-21的调控。5.mi R-21抑制剂联合尼妥珠单抗治疗胶质瘤效果优于单药治疗。6.生物信息学结果显示HOTAIR调节的基因主要参与细胞周期的调控。7.EZH2抑制剂DZNep能模拟si RNA HOTAIR的功能,将胶质瘤细胞周期阻滞在G1期。8.HOTAIR敲低的细胞系中过表达其5’结构域能部分的回复受si RNA HOTAIR引起的细胞周期抑制作用。9.胶质瘤颅内模型证实siRNA HOTAIR能抑制胶质瘤的生长。结论:1.mi R-21通过靶定VHL/β-catenin和PPARα/AP-1调节EGFR/AKT信号传导通路。2.EGFR和mi R-21之间存在反馈调节环路。3.体内外研究显示联合尼妥珠单克隆抗体和mi R-21抑制剂治疗胶质瘤优于单药治疗。4.Lnc RNA HOTAIR调控的基因主要参与细胞周期过程;HOTAIR主要通过其5’结构域和PRC2复合物的结合调节胶质瘤细胞周期进展。5.敲低HOTAIR的表达能在体内抑制胶质瘤的恶性增殖。
[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.

【学位授予单位】:天津医科大学
【学位级别】:博士
【学位授予年份】:2015
【分类号】:R739.4

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