LRIG2基因全长及胞外段抑制胶质瘤细胞系U87凋亡的机制

Full length and ectodomain of LRIG2 overexpression inhibits the apoptosis of glioma cell line U87:analysis of the mechanism

目的研究富含亮氨酸重复序列免疫球蛋白样蛋白2(LRIG2)基因全长及胞外段抑制胶质瘤细胞系U87凋亡的机制。方法将LRIG2全长(LRIG2),LRIG2胞外段(LRIG2ecto)及空白对照(Con)慢病毒表达载体分别感染U87,筛选获得稳定细胞株;流式细胞术检测细胞凋亡率及细胞线粒体膜电位;Western blotting检测凋亡相关蛋白及信号通路蛋白表达。结果 LRIG2及LRIG2ecto过表达组细胞凋亡率分别为2.86%±0.30%和4.04%±0.59%,明显低于对照组5.90%±0.45%(P<0.05);其线粒体膜电位分别是对照组的(2.77±0.25)倍和(2.33±0.17)倍,较对照组明显升高(P<0.01);其凋亡相关蛋白Bcl-2/Bax比值分别是对照组的(1.45±0.09)倍和(1.35±0.08)倍,较对照组明显升高(P<0.01);其表皮生长因子受体(EGFR),磷酸化EGFR(P-EGFR)及其下游的磷酸化蛋白激酶B(p Akt)均较对照组明显增加。结论 LRIG2基因全长及胞外段均可激活EGFR信号通路,抑制胶质瘤细胞凋亡。

Objective The effects of the full length and ectodomain of Leucine-rich repeats and immunoglobulin-like domains-2（ LRIG2） overexpression on the apoptosis of glioma cell line U87 and the underlying mechanism are discussed. Methods The lentiviral vectors expressing the full length and ectodomain of LRIG genes were transfected into glioma cell line U87,respectively. The annexin v-fluorescein isothiocyanate /propidium iodide（ Annexin V-FITC / PI） double labeling flowcytometry was used to detect the apoptotic rates and the 5,5＇,6,6＇-tetrachloro-1,1＇,3,3＇-tetraethyl-benzimidazolylcarbocyanine iodide（ JC-1） labeling flowcytometry was used to detect the mitochondria membrane potential. Western blot was used to investigate the expression of proteins. Results The apoptotic rates of LRIG2 and LRIG2 ecto over-expressing groups were 2. 86% ± 0. 30% and 4. 04% ± 0. 59%,respectively,which were significantly lower than those of control group（5. 90% ± 0. 45%）. Compared to the cells of control group,the mitochondria membrane potentials of LRIG2 and LRIG2 ecto over-expressing cells were significantly increased to（2. 77 ± 0. 25） fold and（2. 33 ±0. 17） fold,respectively. The ratios of Bcl-2 / Bax apoptotic proteins of LRIG2 and LRIG2 ecto groups were significantly increased to（1. 45 ± 0. 09） fold and（1. 35 ± 0. 08） fold,respectively. The expressions of epidermal growth factor receptor（ EGFR）,phospho-EGFR and the downstream phospho-AKT and-ERK of LRIG2 and LRIG2 ecto over-expressing cells were all remarkably increased. Conclusion Overexpressions of LRIG2 and LRIG2 ecto both activate the EGFR signaling pathway and inhibit the apoptosis of the glioma cells.