【作者】 王宝峰；

【导师】 雷霆；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分富含亮氨酸重复序列免疫球蛋白2-胶质瘤细胞表皮生长因子受体信号网络新的调控靶点目的确定富含亮氨酸重复序列免疫球蛋白2(LRIG2)是胶质瘤细胞系GL15中EGFR信号通路新的调控靶点。方法培养胶质瘤细胞系GL 15细胞，经表皮生长因子(EGF)100ng／ml，AG 1478 10μM，放线菌酮(CHX)10μg／ml体外干预后，逆转录聚合酶链反应(RT-PCR)和Western Blot测定LRIG2 mRNA和蛋白表达的时间效应变化。结果RT-PCR结果显示随着EGF作用时间的延长，LRIG2 mRNA先上升后恢复至正常水平。Western blot结果显示EGF作用后，表皮生长因子受体(EGFR)，磷酸化EGFR(pEGFR)均先上升后下降，LRIG2蛋白先下降，在30min时上升，120min下降至原水平的50％。CHX作用1．5h后，再加入EGF刺激，可见LRIG2蛋白60min降至原水平的50％。而AG1478作用1h后，再用EGF刺激，EGFR未受明显影响，pEGFR被明显抑制，LRIG2蛋白水平变化不明显。结论EGFR活化后可导致LRIG2 mRNA表达上升，LRIG2蛋白合成增加。LRIG2为胶质瘤细胞表皮生长因子受体信号网络一个新的调控靶点。第二部分LRIG2基因短发夹RNA表达稳定株的建立及下调LRIG2对EGFR信号通路的影响目的构建针对LRIG2基因的短发夹RNA(shRNA)的表达载体，建立稳定转染的细胞株，观察目的基因表达的变化及其对EGFR信号通路的影响。方法根据GenBank中LRIG2基因序列设计2条RNA干扰序列，命名LRIG2-shRNA1、LRIG2-shRNA2，同时设计1条非特异性序列作为阴性对照。据此设计合成各自的寡核苷酸链，退火后连接入pGenesi12载体，转化扩增后进行序列测定。用不同浓度的G418作用于GL15细胞，得到G418对于GL15细胞的筛选浓度。3种重组表达载体转染胶质瘤细胞系GL15细胞，用G418筛选后挑单克隆后扩增获得稳定株。逆转录RT-PCR和Western印迹法分别在mRNA和蛋白水平上检测LRIG2的表达。用表皮生长因子(EGF)100ng／ml体外干预得到的稳定细胞株，Western Blot测定EGFR和磷酸化EGFR水平的变化。结果3种重组表达载体(pGenesil2-LRIG2-shRNA1、pGenesil2-LRIG2-shRNA2和pGenesil2-negative shRNA)经限制性酶切及DNA测序分析证明序列插入正确。G418对于GL15细胞的筛选浓度为600μg／ml，筛选出稳定转染三种质粒的GL15细胞，转染pGenesil2-LRIG2-shRNA2组细胞LRIG2 mRNA和蛋白表达明显低于转染pGenesil2-negative shRNA组，pGenesil2-LRIG2-shRNA1组LRIG2蛋白无明显变化。EGF刺激5分钟和30分钟后，pGenesil2-LRIG2-shRNA2组细胞EGFR和pEGFR蛋白水平均低于对照组。结论成功构建了针对LRIG2基因的shRNA表达载体(pGenesil2-LRIG2-shRNA2)，转染细胞后可抑制LRIG2基因表达，LRIG2与LRIG1作用相反，下调LRIG2可减少EGF诱导的EGFR磷酸化，促进EGFR降解。第三部分RNA干扰LRIG2后对胶质瘤细胞生长、细胞周期、凋亡、黏附和侵袭能力的影响目的研究RNA干扰LRIG2基因表达后对胶质瘤细胞生长、细胞周期、凋亡、黏附和侵袭能力的影响。方法MTT法检测稳定转染对照组和siRNA组细胞的增殖，流式检测细胞周期和细胞凋亡，细胞黏附实验检测细胞黏附能力，Transwell检测细胞侵袭能力，免疫细胞化学检测PCNA阳性细胞和LRIG2的细胞定位。结果MTT结果显示siRNA组细胞增殖率低于对照组。对照组PCNA阳性细胞率为72％，siRNA组为16％(p＜0．01)。细胞周期分析表明对照组G0／G1期，S期和G2／M期分别为60．18％±5．00％，24．22％±5．37％和15．6％±1．54％，siRNA组为82．40％±2．47％，9．41％±1．87％和8．17％±1．01％(p＜0．01)，siRNA组自发性凋亡增加3倍。LRIG2 RNA干扰组黏附和侵袭能力高于对照组。LRIG2在GL15细胞中主要分布于胞浆。结论RNA干扰GL15细胞LRIG2表达后，抑制细胞增殖和使细胞周期阻滞在G0／G1期，凋亡增加，黏附和侵袭能力增强。下调LRIG2可抑制胶质瘤细胞的生长，有望成为胶质瘤分子治疗的靶点。更多还原

【Abstract】 PartⅠLRIG2—the New Transcriptional Regulatory Target ofEpidermal Growth Factor Receptor(EGFR) Signaling Network inHuman Glioma CellObjective To confirm that LRIG2 is the new regulatory target of epidermal growth factorreceptor(EGFR) signaling network in human glioma cell line GL15.Methods After the human glioma cell line GL15 was exposed to the concentrations of EGF100ng/ml,AG1478 10μM and Cycloheximide 10μg/ml in vitro,changes of mRNA andprotein levels of LRIG2 were measured by reverse transcriptional—polymerase chainreaction(RT-PCR) and Western Blotting.Results The levels of LRIG2 mRNA in GL 15 cell rised after 15 minutes of EGFstimulation,the protein levels of LRIG2 rised after 30 minutes of EGF sitmulation.Cycloheximide and AG1478 were added before the exposure of EGF at designed time on GL15,the protein levels of LRIG2 weren’t change significantly.Conclusion The activation of EGFR could increase the expression of mRNA and protein ofLRIG2.LRIG2 is the new transcriptional regulatory target of EGFR signaling network inhuman glioma cell.PartⅡEstablishment of Stably Transfected Cell Clone ExpressingLRIG2 Specific Short-hairpin RNA and the Research on Effect ofDownregulation of LRIG2 on EGFR Signaling PathwayObjective To construct effective short-hairpin RNA(shRNA) expression vector encodingshRNA targeting LRIG2 gene and to establish the stably transfected cell clone.Toinvestigate the effect ofdownregulation of LRIG2 on EGFR signaling pathway.Methods Design and synthesize two shRNAs sequences based on the sequence of LRIG2mRNA in the GenBank and one scrambled shRNA sequence as negative control.Thesynthesized sequences were cloned into shRNA expression vector pGenesil2.Accordingthe fatal dose of G418 to GL15 cell,the selection concentration of G418 for GL15 cell wasdetermined.The three shRNA vectors were transfected into GL 15 by Metafectinerespectively.The stably transfected cell clones were obtained after the transfected cell werecultured in medium containing G418.Reverse transcriptase-polymerase chain reaction(RT-PCR) and Western Blotting were performed to examine the inhibitory effect on theRNA level and protein level of LRIG2.After cell stimulated by EGF,Western Blotting wasperformed to detect the levels of EGFR and pEGFR protein.Results The recombinant plasmids containing shRNA were analysized by doubleendonuclease digestion and DNA sequencing.The selective concentration of G418 forGL15 cell was 600μg/ml.LRIG2 expression was significantly down-regulated by siRNA as validated by RT-PCR and Western Bloting.After 30 min of EGF treatment,there was verylittle EGFR detected in the LRIG2-siRNA2 cells.The relative expression level of EGFRwas 0.16 in LRIG2-siRNA2 cell and 0.62 in control.After 5 min and 30 min of EGFstimulation,the relative expression level of pEGFR increased to 5.5 and 4.2 in LRIG2siRNA2 cells respectively,while relative expression level of pEGFR only increased to 3.8and 2.6 in control cells respectively.Conclusion RNA interfering(RNAi) mediated by the shRNA expression vector couldsignificantly down-regulate the expression of LRIG2 in glioma cell line GL15.The stabletransfected cell clone was obtained for further study.Know-down of LRIG2 could decreasethe level of EGFR and inhibit the phosphorylation of EGFR in GL15 cell.PartⅢThe Research on Effect of LRIG2 on the Proliferation,CellCycle,Apotosis,Adhesion and Invasion of Human Glioma Cells byRNA InterferenceObjective To determine the effects of LRIG2 on the proliferation,cell cycle,apotosis,adhesion and invasion of glioma cells by RNA interference.Methods The growth curves were determined by the methyl thiazolyl tetrazolium(MTT)assay.Cell cycles and apoptosis were analyzed by flow cytometry.The ability of adhesionand invasion were measured by Cell-matrix adhesion assay and Transwell chamber assay.The staining of PCNA and localization of LRIG2 were performed byimmunocytochemistry.Results The LRIG2-siRNA cell had less proliferation rate than control cell.The PCNAprotein was less stained cell for LRIG2-siRNA group than that in the control After silencingLRIG2 expression,most cells accumulated at G0/G1(p＜0.01),and the proportionof LRIG2-siRNA cells in S and G2/M phase decreased(p＜0.01).Down-regulation of LRIG2 increased the level of spontaneous apoptosis about three-fold compared to control(p＜0.01).The capabilities of adhesion and invasion were enhanced after knockdown ofLRIG2.LRIG2 was localized in the cytoplasmic area.Conclusions Down-regulation of LRIG2 decreased proliferation;G0/G1 arrest;increasedspontaneous apoptosis;enhanced cell adhesion and increased invasion capability of GL15cells in vitro.These findings validate the attractiveness of LRIG2 as a target in gliomatherapy.更多还原