FHL2沉默对骨肉瘤U2OS细胞增殖与凋亡的影响  

Effects of FHL2 silence on the proliferation and apoptosis of osteosarcoma U2OS cells and its mechanism

作　　者：卢文婷 刘爽[1] 李赛赛 刘佳[1] 徐颖茜[1] 王建祥[1] 王敏[1] 

机构地区：[1]中国医学科学院北京协和医学院实验血液学国家重点实验室,天津300020

出　　处：《临床肿瘤学杂志》2018年第1期1-6,共6页Chinese Clinical Oncology

基　　金：中国高校基本科研业务费专项资金资助项目(3332016095);中国医学科学院医学与健康科技创新工程资助项目(2016-I2M-1-001)

摘　　要：目的探讨沉默FHL2表达对骨肉瘤U2OS细胞的增殖和凋亡的影响及其相关机制的研究。方法通过构建FHL2的shRNA干扰载体p LKO.1,制备慢病毒并感染U2OS细胞,分为sh FHL2目的基因干扰组(sh FHL2)和无关序列对照组(SCR);感染96 h后采用实时定量PCR(QPCR)和Western blotting检测两组FHL2 mRNA和蛋白水平。采用MTT方法检测细胞增殖,流式细胞术分析细胞凋亡和细胞周期的变化,Western blotting检测FHL2干扰后U2OS细胞相关信号通路的蛋白表达变化。利用双荧光素酶报告基因系统检测FHL2对p53下游基因转录调控的影响。结果与SCR组比较,sh FHL2组感染96 h后FHL2 mRNA和蛋白水平均降低(P<0.05)。sh FHL2组感染24、48、72、96 h的细胞增殖比分别为1.73±0.08、2.49±0.38、3.26±0.45和4.28±0.29,其中96 h的细胞增殖比低于SCR组(P<0.05);sh FHL2组感染96 h的细胞凋亡率为(17.55±1.05)%,高于SCR组的(7.73±1.12)%,差异有统计学意义(P<0.05);sh FHL2组感染96 h的G_0/G_1期细胞为(68.18±0.78)%,高于SCR组的(59.73±2.28)%,差异有统计学意义(P<0.05)。干扰FHL2表达能够显著上调p53、p21和Bax的表达水平并能增强p53对Bax启动子的促转录活性。结论沉默FHL2表达可明显抑制骨肉瘤U2OS细胞增殖并促进细胞凋亡,使细胞阻滞于G_0/G_1期;FHL2介导了p53对Bax启动子的转录调节作用。FHL2可能会成为新的肿瘤治疗靶点,其机制与p53/Bax和p53/p21通路相关。Objective To explore the effect of silencing the expression of four and a half LIM domains protein 2( FHL2) on the proliferation and apoptosis of osteosarcoma U2OS cells and its mechanism by shRNA. Methods FHL2 was knocked down in U2OS cells by constructing the shRNA interference carrier PLKO.1 of FHL2. The lentivirus was prepared and infected with U2OS cells,which were divided into sh FHL2 target gene interference group( sh FHL2) and unrelated sequence control group( SCR). At 96 h after transfection,real-time quantitative PCR( QPCR) and Western blotting were used to detect the mRNA and protein levels of FHL2 in two groups. MTT assay and flow cytometry were performed to detect the proliferation,cell cycle distribution and apoptotic rate of U2OS cells. Western blotting was used to analyze the level of cell cycle-and apoptotic-related proteins. Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax when FHL2 was overexpressed or was knocked down. Results At 96 h after transfection,the mRNA and protein levels of FHL2 in sh FHL2 group were significantly lower than those in SCR group( P<0. 05). The proliferative ratios of sh FHL2 group at 24,48,72 and 96 h after transfection were 1. 73±0. 08,2. 49±0. 38,3. 26±0. 45 and 4. 28±0. 29,and the data of 96 h was lower than SCR group( P<0. 05). The apoptotic rate of sh FHL2 group at 96 h after transfection was( 17. 55±1. 05) %,higher than( 7. 73±1. 12) % of SCR group( P<0. 05). The proportion of sh FHL2 group cells in G_0/G_1 phase at 96 h after transfection was( 68. 18±0. 78) %,higher than( 59. 73± 2. 28) % of SCR group,and the difference was statistically significant( P <0. 05). Western blotting showed that the level of cell cycle-related protein p21 and pro-apoptotic protein p53 and Bax increased. Dual luciferase assay suggested that p53 could increase the transcriptional activity of Bax,furthermore,when FHL2 was knocked down at the same time,the transcriptional activity of p53 was enhanced. Conclusion Cell proliferation reduc

关 键 词：骨肉瘤 FHL2 U2OS细胞株 增殖 凋亡 

分 类 号：R738.6[医药卫生—肿瘤]