RNA干扰LRIG1基因对人胶质母细胞瘤GL15细胞系生物学行为的影响及机制研究
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摘要
第一部分LRIG1基因特异性RNA干扰表达载体的构建、鉴定和稳定株的筛选
目的构建针对LRIG1(leucine-rich repeats and immunoglobulin-like domains 1,LRIG1)基因的特异性RNA干扰质粒，稳定转染人胶质母细胞瘤GL15细胞系，观察其对目的基因LRIG1表达的影响，为探讨LRIG1基因沉默对人脑胶质瘤细胞生物学行为的调控奠定基础。
方法根据GenBank提供的LRIG1基因序列设计2条RNA干扰序列，命名为LRIG1-shRNA1、LRIG1-shRNA2，并设计1条非特异性序列作为阴性对照，命名为pGenesil2-negative shRNA。合成各自的寡核苷酸链，退火后与pGenesil2质粒载体连接，转化扩增后测序。用不同浓度的G418作用于GL15细胞确定G418对GL15的筛选浓度。将3种重组表达载体转染GL15细胞，G418筛选后挑单克隆并扩增获得稳定株。Western印迹法在蛋白水平上检测LRIG1的表达。
结果重组pGenesil2-LRIG1-shRNA质粒经限制性酶切及DNA测序分析证明序列插入正确。G418对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-LRIG1-shRNA1组细胞LRIG1蛋白表达明显低于转染pGenesil2-negative shRNA组。
结论成功构建了针对LRIG1基因的特异性shRNA表达载体(pGenesil2-LRIG1-shRNA1)，转染细胞后可抑制LRIG1基因表达，为下一步研究其功能奠定基础。
第二部分RNA干扰LRIG1基因对胶质母细胞GL15细胞系细胞周期、凋亡和增殖的影响
目的研究RNA干扰LRIG1基因表达后对人胶质母细胞瘤GL15细胞系细胞周期、凋亡和增殖的影响。
方法在第一部分构LRIG1基因的特异性RNA干扰质粒的基础上，构建针对LRIG1基因的干扰片段(RNAi组)及非特异性的对照片段(negative组)分别转染GL15细胞系，G418(600μg／ml)筛选出稳定株，流式细胞仪检测对照组和实验组细胞的细胞周期变化，Annexin V-FITC／PI双标记流式细胞仪分析细胞凋亡，MTT法检测LRIG1表达下调对胶质瘤细胞增殖的影响。
结果流式细胞仪分析显示，实验组的G2／M期细胞百分率比对照组明显增加(p＜0.01)；细胞增殖指数显著增加(p＜0.01)；实验组GL15细胞在LRIG1基因表达下调后早期凋亡的细胞明显减少(p＜0.01)；MTT结果显示干扰组细胞增殖率高于对照组(p＜0.01)。
结论GL15细胞经RNA干扰后LRIG1表达明显下调，对GL15细胞生长及抑制细胞凋亡有促进作用。
第三部分RNA干扰LRIG1基因后对胶质母细胞瘤EGFR表达的影响及分子机制的研究
目的探讨富含亮氨酸重复序列免疫球蛋白样蛋白1(LRIG1)基因与表皮生长因子受体(EGFR)在人脑胶质瘤细胞中的相互作用及其对肿瘤细胞生长影响的机制。方法用免疫共沉淀法检测人脑胶质瘤细胞系GL15中LRIG1与EGFR的相互作用。Western-blot法检测LRIG1表达的改变及其对EGFR、Akt、MAPK表达的影响。
结果免疫共沉淀法表明胶质瘤细胞系GL15中LRIG1与EGFR存在相互作用。Western-blot结果显示RANi组LRIG1蛋白水平与阴性对照组相下降了47.9％(p＜0.01)，同时RANi组细胞EGFR、pAkt、pMAPK蛋白水平与阴性对照组相分别上升了57.1％(p＜0.01)、43.6％(p＜0.01)、52.3％(p＜0.01)。
结论干扰LRIG1表达可增强EGFR信号通路，导致肿瘤细胞增殖加速。LRIG1水平有望成为判断胶质瘤恶性程度及预后的参考标准之一，并为其治疗提供手段。
PartⅠConstruction of LRIG1 Specific RNAi Expressing Vector andScreening of Stably Transfected Cell Clon
Objective To construct eukaryotic expression vector encoding RNA interferencesequences specific for LRIG1 gene, to screen the stably transfected cell clone, and toobserve its effect on the expression of LRIG1.
Methods Designed two shRNAs sequences based on the sequence of LRIG1 mRNA in theGenBank and one scrambled shRNA sequence as negative control. The synthesizedsequences were inserted into shRNA expression vector pGenesil2 and sequenced. TheshRNA vectors were transfected into GL15 by Metafectine. The stably transfected cellclones were obtained after being screened with G418.Western Blotting was performed toexamine the inhibitory effect at the protein level.
Results The recombinant plasmids containing shRNA were analysized by restrictionenzyme digest analysis and DNA sequencing. The screening concentration of G418 toGL15 cell was 600μg/ml. LRIG1 expression was significantly down-regulated by siRNA as validated by Western Bloting.
Conclusion RNA interfering (RNAi) mediated by the shRNA expression vector couldsignificantly down-regulate the expression of LRIG1 in glioma cell line GL 15. The stabletransfected cell clone was obtained for further study.
PartⅡThe effect of Down-regulation of LRIG1 Gene Expression onCell Cycle,Apoptosis and Proliferation inGlioblastoma Cell Line GL15
Objective To investigate the effect of down-regulation of LRIG1 gene expression on cellcycle, apoptosis and proliferation in glioblastoma cell line GL 15.
Methods The cell cycle in control and experimental group was detected by flowcytometry. Annexin V-FITC/PI double-labeled flow cytometry analyzed apoptosis. Methylthiazolyl tetrazolium (MTT)detected cell proliferation.
Results Flow cytometry analysis showed that the G2 / M phase cell percentage inexperimental group was higher than control group significantly. Cell proliferation indexincreased significantly (p＜0.01); In shRNAi group, GL15 cells in early apoptotic decreasedsignificantly after down regulation of LRIG1 (p＜0.01) The role of LRIG1 onanti-apoptotic is remarkably (p＜0.01); results of MTT showed that cell proliferation ratein interfered group was higher than control (p＜0.01 ) .
Conclusion The expression of LRIG1 is down regulated significantly in GL15 cell afterRNA interference, which enhances cell growth.
PartⅢThe Effect of RNA interference of Gene LRIG1 on EGFRExpression in Glioblastoma Cell Line(GL15) andthe Molecular Mechanisms
Objective To explore the interaction between the leucine-rich repeat Ig-like protein 1(LRIG1) and epidermal growth factor receptor (EGFR) in human glioma cells and theeffect of LRIG1 on the tumor cell growth.
Method The interaction of LRIG1 and EGFR in human glioma cell line GL15 wasconfirmed by co-immunoprecipitation. Western-blot was used to analysis the level ofEGFR、pAkt、pMAPK protein after silencing LRIG1.
Results Immune co-precipitation method showed that the GL15 glioma cell lines inexistence LRIG1 interaction with EGFR. The LRIG1 protein level inpGenesil2-LRIG1-shRNA (siRNA) transfected cells was significantly silenced by47.9%and the EGFR,Akt,MAPK protein level in it rised by 57.1%,43.6 %,52.3 %.
Conclusion Knowdown of LRIG1 could enhance the functions of EGFR signalingpathway and promote tumor proliferation. LRIG1 protein level could be one index to adjustmalignant degree and prognose of glioma, and provides a new treatment for glioma
目的构建针对LRIG1(leucine-rich repeats and immunoglobulin-like domains 1,LRIG1)基因的特异性RNA干扰质粒，稳定转染人胶质母细胞瘤GL15细胞系，观察其对目的基因LRIG1表达的影响，为探讨LRIG1基因沉默对人脑胶质瘤细胞生物学行为的调控奠定基础。
方法根据GenBank提供的LRIG1基因序列设计2条RNA干扰序列，命名为LRIG1-shRNA1、LRIG1-shRNA2，并设计1条非特异性序列作为阴性对照，命名为pGenesil2-negative shRNA。合成各自的寡核苷酸链，退火后与pGenesil2质粒载体连接，转化扩增后测序。用不同浓度的G418作用于GL15细胞确定G418对GL15的筛选浓度。将3种重组表达载体转染GL15细胞，G418筛选后挑单克隆并扩增获得稳定株。Western印迹法在蛋白水平上检测LRIG1的表达。
结果重组pGenesil2-LRIG1-shRNA质粒经限制性酶切及DNA测序分析证明序列插入正确。G418对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-LRIG1-shRNA1组细胞LRIG1蛋白表达明显低于转染pGenesil2-negative shRNA组。
结论成功构建了针对LRIG1基因的特异性shRNA表达载体(pGenesil2-LRIG1-shRNA1)，转染细胞后可抑制LRIG1基因表达，为下一步研究其功能奠定基础。
第二部分RNA干扰LRIG1基因对胶质母细胞GL15细胞系细胞周期、凋亡和增殖的影响
目的研究RNA干扰LRIG1基因表达后对人胶质母细胞瘤GL15细胞系细胞周期、凋亡和增殖的影响。
方法在第一部分构LRIG1基因的特异性RNA干扰质粒的基础上，构建针对LRIG1基因的干扰片段(RNAi组)及非特异性的对照片段(negative组)分别转染GL15细胞系，G418(600μg／ml)筛选出稳定株，流式细胞仪检测对照组和实验组细胞的细胞周期变化，Annexin V-FITC／PI双标记流式细胞仪分析细胞凋亡，MTT法检测LRIG1表达下调对胶质瘤细胞增殖的影响。
结果流式细胞仪分析显示，实验组的G2／M期细胞百分率比对照组明显增加(p＜0.01)；细胞增殖指数显著增加(p＜0.01)；实验组GL15细胞在LRIG1基因表达下调后早期凋亡的细胞明显减少(p＜0.01)；MTT结果显示干扰组细胞增殖率高于对照组(p＜0.01)。
结论GL15细胞经RNA干扰后LRIG1表达明显下调，对GL15细胞生长及抑制细胞凋亡有促进作用。
第三部分RNA干扰LRIG1基因后对胶质母细胞瘤EGFR表达的影响及分子机制的研究
目的探讨富含亮氨酸重复序列免疫球蛋白样蛋白1(LRIG1)基因与表皮生长因子受体(EGFR)在人脑胶质瘤细胞中的相互作用及其对肿瘤细胞生长影响的机制。方法用免疫共沉淀法检测人脑胶质瘤细胞系GL15中LRIG1与EGFR的相互作用。Western-blot法检测LRIG1表达的改变及其对EGFR、Akt、MAPK表达的影响。
结果免疫共沉淀法表明胶质瘤细胞系GL15中LRIG1与EGFR存在相互作用。Western-blot结果显示RANi组LRIG1蛋白水平与阴性对照组相下降了47.9％(p＜0.01)，同时RANi组细胞EGFR、pAkt、pMAPK蛋白水平与阴性对照组相分别上升了57.1％(p＜0.01)、43.6％(p＜0.01)、52.3％(p＜0.01)。
结论干扰LRIG1表达可增强EGFR信号通路，导致肿瘤细胞增殖加速。LRIG1水平有望成为判断胶质瘤恶性程度及预后的参考标准之一，并为其治疗提供手段。
PartⅠConstruction of LRIG1 Specific RNAi Expressing Vector andScreening of Stably Transfected Cell Clon
Objective To construct eukaryotic expression vector encoding RNA interferencesequences specific for LRIG1 gene, to screen the stably transfected cell clone, and toobserve its effect on the expression of LRIG1.
Methods Designed two shRNAs sequences based on the sequence of LRIG1 mRNA in theGenBank and one scrambled shRNA sequence as negative control. The synthesizedsequences were inserted into shRNA expression vector pGenesil2 and sequenced. TheshRNA vectors were transfected into GL15 by Metafectine. The stably transfected cellclones were obtained after being screened with G418.Western Blotting was performed toexamine the inhibitory effect at the protein level.
Results The recombinant plasmids containing shRNA were analysized by restrictionenzyme digest analysis and DNA sequencing. The screening concentration of G418 toGL15 cell was 600μg/ml. LRIG1 expression was significantly down-regulated by siRNA as validated by Western Bloting.
Conclusion RNA interfering (RNAi) mediated by the shRNA expression vector couldsignificantly down-regulate the expression of LRIG1 in glioma cell line GL 15. The stabletransfected cell clone was obtained for further study.
PartⅡThe effect of Down-regulation of LRIG1 Gene Expression onCell Cycle,Apoptosis and Proliferation inGlioblastoma Cell Line GL15
Objective To investigate the effect of down-regulation of LRIG1 gene expression on cellcycle, apoptosis and proliferation in glioblastoma cell line GL 15.
Methods The cell cycle in control and experimental group was detected by flowcytometry. Annexin V-FITC/PI double-labeled flow cytometry analyzed apoptosis. Methylthiazolyl tetrazolium (MTT)detected cell proliferation.
Results Flow cytometry analysis showed that the G2 / M phase cell percentage inexperimental group was higher than control group significantly. Cell proliferation indexincreased significantly (p＜0.01); In shRNAi group, GL15 cells in early apoptotic decreasedsignificantly after down regulation of LRIG1 (p＜0.01) The role of LRIG1 onanti-apoptotic is remarkably (p＜0.01); results of MTT showed that cell proliferation ratein interfered group was higher than control (p＜0.01 ) .
Conclusion The expression of LRIG1 is down regulated significantly in GL15 cell afterRNA interference, which enhances cell growth.
PartⅢThe Effect of RNA interference of Gene LRIG1 on EGFRExpression in Glioblastoma Cell Line(GL15) andthe Molecular Mechanisms
Objective To explore the interaction between the leucine-rich repeat Ig-like protein 1(LRIG1) and epidermal growth factor receptor (EGFR) in human glioma cells and theeffect of LRIG1 on the tumor cell growth.
Method The interaction of LRIG1 and EGFR in human glioma cell line GL15 wasconfirmed by co-immunoprecipitation. Western-blot was used to analysis the level ofEGFR、pAkt、pMAPK protein after silencing LRIG1.
Results Immune co-precipitation method showed that the GL15 glioma cell lines inexistence LRIG1 interaction with EGFR. The LRIG1 protein level inpGenesil2-LRIG1-shRNA (siRNA) transfected cells was significantly silenced by47.9%and the EGFR,Akt,MAPK protein level in it rised by 57.1%,43.6 %,52.3 %.
Conclusion Knowdown of LRIG1 could enhance the functions of EGFR signalingpathway and promote tumor proliferation. LRIG1 protein level could be one index to adjustmalignant degree and prognose of glioma, and provides a new treatment for glioma
引文
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