RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白2后对胶质瘤细胞生长的影响及其机制

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pGenesi12-LRIG2-shRNA(siRNA)及含非特异性shRNA编码序列的对照质粒pGenesi12-scramble-shRNA(scr)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Western blot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LRIG2 mRNA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,LRIG2 mRNA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.

Effects of LRIG2 on proliferation of human glioma cells by RNA interference and the mechanism

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 pGenesi12-LRIG2-shR-NA (siRNA) was transfected into GL15 glioma cells by Metafeetine, and the cells (siRNA) that had a stably suppressive LRIG2 expression were selected by G418.The control cells 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 tet-rasolium (MTT) assay.Cell cycles were analyzed by flow eytometry.Results The LRIG2 mRNA and pro-tein levels in pGenesi12-LRIG2-shRNA (siRNA) transfected cells were significantly lower than in scram-bled shRNA (ser) transfected cells.The EGFR protein level in pGenesil2-LRIG2-ShRNA (siRNA) cell was lower than in ser cells.The siRNA cells had a lower proliferation rate than ser 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 sup-press the proliferation of GL15 cell and arrest the cell cycle in G0/G1 phase.