N-乙酰氨基半乳糖转移酶14通过细胞外信号调节激酶1/2信号通路调控多形性胶质母细胞瘤凋亡的机制

Mechanism of N-acetyl amino galactosyl transferase-14 regulating the apoptosis of glioblastoma through extracellular signal-regulated kinase 1/2 signaling pathway

目的探讨N-乙酰氨基半乳糖转移酶14(GalNAc-T14)在胞内高表达促进多形性胶质母细胞瘤凋亡的机制。方法培养多形性胶质母细胞瘤U87MG细胞株,构建的pcDNA3.1-T14质粒转染U87MG细胞,分为未处理组(Control组)、转染pcDNA3.1(+)空载体组(NC1组)、转染pcDNA3.1-T14组(T组),采用实时定量聚合酶链反应(qPCR)、蛋白质印迹法(Western blot)检测GalNAc-T14、细胞外信号调节激酶1/2(ERK1/2)、B细胞淋巴瘤/白血病-XL(bcl-XL)的表达、ERK1/2的磷酸化水平;采用膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙锭(PI)双染色的方法,在流式细胞仪上检测细胞凋亡情况。应用SPSS 13.0统计软件分析,计量资料均值±标准差(Mean±SD)表示。结果质粒转染前GalNAc-T14、ERK1/2、bcl-XL相对表达量为1,在U87MG细胞内高表达GalNAc-T14相对量为(15393.43±30.35)。qPCR显示ERK1、ERK2、bcl-XL的mRNA相对表达量:NC1组为(0.98±0.12)、(0.91±0.05)、(0.89±0.11),T组为(0.74±0.29)、(0.68±0.31)、(0.68±0.23),T组ERK1/2与抗凋亡蛋白bcl-XL的基因表达水平较对照组下降;Westernblot分析显示:Control组、NC1组、T组ERK1/2的灰度值分别为(85.42±3.11、86.25±4.25、66.36±4.25),抗凋亡蛋白bcl-XL的灰度值为(82.57±3.71、79.52±3.78、46.85±4.52)。通过Annexin V-FITC/PI双染色的方法,在流式细胞仪上检测细胞凋亡显示高表达GalNAc-T14时U87MG细胞株早期凋亡与晚期凋亡的百分数分别为(7.74±1.23)%、(10.08±2.41)%,高于对照组(1.12±0.18)%、(0.51±0.14)%及NC1组(1.37±0.12)%、(0.90±0.08)%,差异有统计学意义(t=3.417,P<0.05)。结论GalNAc-T14通过抑制ERK1/2及bcl-XL的表达促进多形性胶质母细胞瘤细胞的凋亡。

Objective To study the effect of N-acetyl amino galactosyl transferase-14 (GalNAc-T14) on apoptosis of glioblastoma multiforme. Methods The U87MG cell line of glioblastoma multiforme was cultured, and the constructed pcDNA3.1-T14 plasmid was transfected into U87MG cells. The cells were divided into untreated group (control), pcDNA3.1 (+) empty carrier-transfected group (NC1) and pcDNA3.1-T14-transfected group (T). Real-time quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the expression of GalNAc-T14, extracellular signal-regulated kinase 1/2 (ERK1/2), inhibitor of apoptosis protein-XL (bcl-XL) and the phosphorylation level of ERK1/2. Apoptosis was detected by flow cytometry with Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) double staining. Results The relative quality of GalNAc-T14, ERK1/2 and bcl-XL was 1 before transfection, and the relative expression of GalNAc-T14 was (15 393.43±30.35) in U87MG cells. Real-time quantitative PCR showed that the relative mRNA expression of ERK1, ERK2 and bcl-XL in NC1 group was (0.98±0.12),(0.91±0.05) and (0.89±0.11) respectively, and that in T group was (0.74±0.29),(0.68±0.31) and (0.68±0.23) respectively. The expression level of ERK1/2 and anti-apoptotic protein bcl-XL in T group was lower than that in control group. Western blotting analysis showed that the gray values of ERK1/2 in control, NC1 and T groups were (85.42±3.11),(86.25±4.25), and (66.36±4.25), and those of anti-apoptotic protein bcl-XL were (82.57±3.71),(79.52±3.78), and (46.85±4.52), respectively. The percentages of early apoptosis and late apoptosis of U87MG cells was (7.74±1.23)% and (10.08±2.41)%, respectively, when high expression of GalNAc-T14 was detected by Annexin V-FITC/PI double staining, which were higher than in the control group [(1.12±0.18)%,(0.51±0.14)%] and NC1 group [(1.37±0.12)%,(0.90±0.08)%, t=3.417, P<0.05]. Conclusion GalNAc-T14 promotes apoptosis of glioblastoma cells by inhibiting the expression of ERK1/2 and bcl-XL.