膜联蛋白A5对胶质瘤细胞侵袭和迁移的调节作用及机制

Regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanisms

目的研究膜联蛋白A5对胶质瘤细胞侵袭和迁移的调节作用及机制。方法免疫组化法检测100例神经胶质瘤组织和20例正常脑组织中膜联蛋白A5的表达。对人神经胶质瘤细胞系(U251)转染靶向膜联蛋白A5的siRNA(si-Annexin A5组),并通过RT-PCR和蛋白印迹分析验证膜联蛋白A5表达下调。MTT实验和集落形成实验检测U251细胞的增殖能力,流式细胞术和Hoechst 33258染色检测U251细胞凋亡,伤口愈合实验和Matrigel Transwell侵袭实验观察U251细胞的迁移和侵袭能力。蛋白印迹分析U251细胞中Raf、p-Raf、MEK1/2、p-MEK1/2、ERK1/2、p-ERK1/2、c-Myc和E-Cadherin的表达。结果与正常脑组织相比,胶质瘤组织中的膜联蛋白A5的mRNA表达升高了2.45倍,蛋白表达升高了2.87倍(P<0.001);Pearson相关分析显示,随着肿瘤级别的升高,膜联蛋白A5阳性率逐渐升高,并且肿瘤级别与阳性率呈正相关(r=1.000,P<0.001)。与对照组相比,si-Annexin A5组的U251细胞在培养48、72 h的细胞活力分别降低了29.46%和40.43%(P<0.001)。与对照组相比, si-Annexin A5组的集落形成率降低了68.58%,而细胞凋亡率升高了24.41倍(P<0.001);si-Annexin A5组的迁移细胞率降低了65.35%,侵袭细胞率降低了68.80%(P<0.001);si-Annexin A5组的p-Raf、p-MEK1/2、p-ERK1/2和c-Myc的蛋白表达均明显降低(依次降低54.67%、70.37%、60.26%、54.95%),而E-Cadherin升高了3.58倍(P<0.001)。结论下调膜联蛋白A5通过抑制Raf/MEK/ERK信号通路抑制胶质瘤细胞的生长和运动能力,并诱导细胞凋亡。

Objective To investigate the regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanism. Methods The expression of annexin A5 in 100 cases of glioma tissues and 20 cases of normal brain tissues was detected by immunohistochemistry. The expression of annexin A5 was downregulated by transfection with siRNA targeting annexin A5(si-Annexin A5) in human glioma cell line(U251). The expression of annexin A5 was confirmed by RT-PCR and Western blot analysis. The proliferation ability of U251 cells was detected by MTT test and colony formation test, the apoptosis of U251 cells was detected by flow cytometry and Hoechst 33258 staining, and the migration and invasion ability of U251 cells was examined by wound healing test and Matrigel Transwell invasion test. The expressions of Raf, p-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Myc and E-Cadherin in U251 cells were analyzed by Western blot. Results Compared with those of normal brain tissues, the mRNA and protein expression levels of Annexin A5 in glioma tissues increased by 2.45 times and 2.87 times, respectively(P<0.05). Pearson correlation analysis showed that with the increase of tumor grade, the positive rate of Annexin A5 gradually increased,and the tumor grade and positive rate were significantly positively correlated(r=1.000,P=0.000).The cell viability of U251 cells in the si-Annexin A5 group after 48 hand 72 hof culture was significantly reduced by 29.46% and 40.43%,respectively,compared with that in the control group(P<0.05).Compared with the control group,in the si-Annexin A5 group the colony formation rate was reduced by68.58%,while the apoptosis rate was increased by 24.41 times(P<0.05);the cell migration rate and invasion rate were reduced by 65.35%and 68.80%(P<0.05).The protein expression of p-Raf,p-MEK1/2,p-ERK1/2 and c-Myc in the si-Annexin A5 group were significantly reduced by 54.67%,70.37%,60.26%and 54.95%,respectively,and that of E-Cadherin was increased by 3.58 times(P<0.05).Conclusion Downregulation of Annexin A5 inhibits the growth and motility of glioma cells and induces cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway.