RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白3表达对胶质瘤细胞周期和凋亡的影响

Effects of RNAi-mediated gene silencing of LRIG3 expression on the cell cycle and apoptosis of glioma cells

目的探讨RNA干扰富含亮氨酸重复序列免疫球蛋白样蛋白3（LRIG3）基因表达后对胶质瘤细胞生长的影响及其机制。方法设计两对含LRIG3特异性短发夹RNA（shRNA）序列的质粒及含非特异性shRNA的对照质粒，转染胶质瘤细胞系GL15、G418筛选出稳定株，逆转录-聚合酶链反应（RT—PCR）和Westernblot法检测LRIG3表达的改变，流式细胞仪检测各组细胞的细胞周期变化，AnnexinV—FITC／PI双标记分析细胞凋亡。结果实验组细胞LRIG3mRNA和蛋白水平与对照组比较分别下降了52．4％、63．8％和50．9％、67．4％。流式细胞仪分析显示，实验组的G2／M期细胞细胞百分率比对照组明显增加，细胞增殖指数显著增加（P〈0．01）；LRIG3基因表达下调后还具有显著的抗凋亡作用（P〈0．05）。结论LRIG3特异性siRNA可明显抑制LRIG3基因的转录和表达，从而促进GL15细胞的增殖和使细胞周期阻滞在G2／M期并能抑制其凋亡。

Objective To explore the effects of inhibiting the expression of LRIG3 gene on proliferation and apoptosis of human glioma cell line GL15 and explore its possible mechanism by using small interfering RNA （siRNA） targeting LRIG3 gene. Methods Two pairs of pGenesil2-LRIG3-shRNA （ siRNA） were transfected into GL15 glinma cells by Metafectine as experimental groups 1 and 2 ,and the cells that had a stably suppressive LRIG3 expression were selected by G418. The control cells were transfected with negative shRNA. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blot. The apoptosis rate and cell cycle were analyzed by flow cytometry. Results Compared with LRlG3 InRNA expression in the negative shRNA-treated GL15 cells,the transcription after treatment with LR1G3-specific shRNA was silenced by 52.4% and 63.8% ,and the expression of LR1G3 protein was reduced by about 50.9% and 67.4% in experimental groups 1 and 2 respectively. Cell cycle analysis showed that silencing LRIG3 increased the percentage of G2/M phase cells and improved the proliferation index significantly （ P 〈 0.01 ）. Conclusion The siRNA targeting LR1G3 gene can dramatically inhibit RNA transcription and protein expression, then promote the proliferation of GL15 cells and suppress the apoptosis of GL15 cells.