摘要
目的探讨RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白2（LRIG2）基因表达后对胶质瘤细胞生长的影响及其机制。方法用携带U6启动子和LRIG2特异性短发夹RNA（shRNA）序列的质粒载体pGenesil2-LRIG2-shRNA（siRNA）及含非特异性shRNA编码序列的对照质粒pGenesil2-scramble—shRNA（set）转染胶质瘤细胞系GL15、G418（600mg/L）筛选出稳定株，逆转录-聚合酶链反应（RT—PCR）和Westemblot法检测LRIG2和表皮生长因子受体（EGFR）表达的改变。噻唑蓝（MTT）比色法检测稳定转染对照组和实验组细胞的增殖，流式细胞仪检测对照组和实验组细胞的细胞周期改变。结果实验组细胞LRIG2mRNA和蛋白水平与对照组比较分别下降了68．6％和62．3％，EGFR蛋白水平下降了32．6％。MTT结果显示实验组细胞增殖率低于对照组。细胞周期分析表明对照组G0／G1期为（26．72±2．10）％，实验组为（36．58±3．29）％（P〈0．05），LRIG2表达下调后GL15细胞细胞周期部分阻滞在G0／G1期。结论GL15细胞经RNA干扰基因表达后，LRIG2mRNA和蛋白水平明显降低，同时EGFR蛋白水平下降，从而抑制细胞增殖和使细胞周期阻滞在G0／G1期。LRIG2可通过EGFR信号系统影响胶质瘤细胞的生长。
Objective To study the effects of LRIG2 on the proliferation of glioma cells by RNA interference and the mechanisms underlying such effects. Methods The plasmid pGenesil2-LRIG2-shR- NA （siRNA） was transfected into GL15 glioma cells by Metafectine, and the ceils （siRNA） that had a stably suppressive LRIG2 expression were selected by G418. The control ceils were transfected with scram- bled shRNA （scr）. The changes in LRIG2, and epidermal growth factor receptor （EGFR） protein levels were measured by RT-PCR and Western blot. Growth curves were determined by the methyl thiazolyl tetrazolium （MTF） assay. Cell cycles were analyzed by flow cytometry. Results The LRIG2 mRNA and protein levels in pGencsil2-LRIG2-shRNA （siRNA） transfected cells were significantly lower than in scrambled shRNA （scr） transfected cells. The EGFR protein level in pGenesil2-LRIG2-ShRNA （siRNA） cell was lower than in scr cells. The siRNA cells had a lower proliferation rate than scr cells. Cell cycle analysis showed that silencing LRIG2 caused G0/G1 cell cycle arrest. Conclusion Silencing LRIG2 expression could result in degradation of EGFR, then affect the downstream pathway of EGFR signaling, further suppress the proliferation of GL15 cell and arrest the cell cycle in G0/G1 phase.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第3期375-377,共3页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项目（30500521）
