转录因子E2F1过表达对脑胶质瘤U251细胞辐射敏感性的影响  

Effects of over-expression of E2F transcription factor 1 on radiosensitivity glioma U251 cells

作　　者：黄从刚[1] 罗明[1] 陈谦学[2] 王远[1] 黄乔春[1] 周洁 王兴弯 罗志华[1] 段发亮[1] Huang Conggang;Luo Ming;Chen Qianxue;Wang Yuan;Huang Qiaochun;Zhou Jie;Wang Xingwan;Luo Zhihua;Duan Faliang(Department of Neurosurgery,Wuhan No.1 Hospital,Wuhan 430022,China;Department of Neurosurgery,Renmin Hospital of Wuhan University,Wuhan 430060,China;Department of Interventional Radiology,Wuhan NO.1 Hospital,Wuhan 430022,China)

机构地区：[1]武汉市第一医院神经外科,武汉430022 [2]武汉大学人民医院神经外科,武汉430060 [3]武汉市第一医院介入放射科,武汉430022

出　　处：《中华行为医学与脑科学杂志》2023年第3期218-224,共7页Chinese Journal of Behavioral Medicine and Brain Science

基　　金：湖北省卫生健康委科研基金资助面上重点项目(WJ2019H331);武汉市卫生健康委科研基金资助面上重点项目(WX19A16)。

摘　　要：目的:探讨过表达E2F转录因子1(E2F transcription factor 1,E2F1)对胶质瘤细胞U251细胞增殖、侵袭、凋亡以及辐射敏感性的影响。方法:实时荧光定量PCR(qRT-PCR)检测E2F1 mRNA在胶质瘤细胞LN18、SW1088、U251和正常人脑神经胶质细胞HEB中的差异表达情况。构建稳定过表达E2F1的质粒并转染至U251细胞。qRT-PCR和免疫印迹实验(Western blot)检测对照组和E2F1过表达组中E2F1、垂体肿瘤转化基因1(PTTG1)、原癌基因C-Myc、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA和蛋白的表达。将U251细胞分为对照组(无X射线照射)、照射组(6 Gy X射线照射)和照射+E2F1过表达组(转染E2F1后,6 Gy X射线照射)。细胞计数试剂盒CCK-8法检测细胞增殖能力,细胞侵袭迁移实验(Transwell小室)检测细胞侵袭和迁移能力,流式细胞术检测细胞凋亡和细胞周期。使用GraphPad Prism 8.0进行数据分析,统计方法采用单因素方差分析、独立样本t检验。结果:qRT-PCR检测结果显示,E2F1 mRNA在LN18(4.04±0.29)、SW1088(3.19±0.16)、U251(4.66±0.20)、HEB(1.02±0.07)细胞中的表达水平差异有统计学意义(F=201.92,均P<0.05),LN18、SW1088、U251细胞中E2F1 mRNA表达水平高于HEB细胞(q=27.00,19.40,40.32,均P<0.05)。成功构建稳定过表达E2F1质粒的U251细胞后,qRT-PCR和Western blot检测结果显示:E2F1过表达组细胞中E2F1、PTTG1、C-Myc、Bcl-2 mRNA和蛋白水平均高于对照组(t=77.16,57.88,4.63,51.13,7.50,70.85,8.38,48.81),差异有统计学意义(均P<0.05),Bax mRNA(0.20±0.01)和蛋白水平(0.66±0.01)低于对照组((1.00±0.02)、(0.94±0.01)),均差异有统计学意义(t=1.74,54.65,均P<0.05)。经过X射线照射(6 Gy)后,CCK8检测结果显示:3组细胞在24、48、72、96 h的细胞增殖能力均差异有统计学意义(F=95.41,187.53,1158.49,7883.78,均P<0.05)。照射组24、48、72、96 h时的细胞增殖能力低于对照组(q=19.51,27.20,66.60,174.9,均P<0.05),照射+E2F1过表达组24、48、72、96 h的细�Objective To investigate the effects of over-expression of E2F transcription factor 1(E2F1)on proliferation,invasion,apoptosis and radiosensitivity of glioma cell U251.Methods Real-time quantitative PCR(qRT-PCR)were used to detect the differential expression of E2F1 mRNA in glioma cells LN18,SW1088,U251 and normal brain glial cells.The stable over-expression of E2F1 plasmid was constructed and transfected into U251 cells.qRT-PCR and Western blot test were used to detect the expression of E2F1,pituitary tumor transforming gene 1(PTTG1),C-Myc,B-cell lymphoma-2(Bcl-2),Bcl2-associated X(Bax)mRNA and protein expression in the control group and E2F1 over-expression group.U251 cells were divided into control group(no X-ray irradiation),irradiation group(6 Gy dose of X-ray),and irradiation+E2F1 over-expression group(transfected with E2F1 first,then irradiated by 6 Gy of X-ray).Cell proliferation ability was detected by cell counting Kit-8(CCK-8)cell viability detection reagent,and cell invasion and migration ability were detected by Transwell chamber.Apoptosis and cell cycle were detected by flow cytometry.GraphPad Prism 8.0 was used for data analysis.The statistical methods were one-way ANOVA and independent sample t-test.Results qRT-PCR showed that there was statistical difference in the mRNA levels of E2F1(F=201.92,P<0.05)in different cell lines.The expression levels of E2F1 mRNA in LN18(4.04±0.29),SW1088(3.19±0.16)and U251(4.66±0.20)cells were higher than those in HEB(1.02±0.07)cells(q=27.00,19.40,32.52,all P<0.05).After successfully constructing U251 cells with stable over-expression of E2F1 plasmid,qRT-PCR and Western blot detection results showed that:the mRNA and protein levels of E2F1,PTTG1,C-Myc and Bcl-2 in E2F1 over-expression group were higher than those in control group(t=77.16,57.88,4.63,51.13,7.50,70.85,8.38,48.81,all P<0.05).Bax mRNA(0.20±0.01)and protein(0.66±0.01)levels were lower than those in control group((1.00±0.02),(0.94±0.01)),and the differences were statistically significant(t=1.74,54.6

关 键 词：E2F转录因子1 胶质瘤U251 辐射敏感性 

分 类 号：R739.41[医药卫生—肿瘤]