肾母细胞瘤过表达基因对人骨肉瘤143B细胞增殖、凋亡和迁移的影响  

Effects of nephroblastoma overexpressed gene on proliferation, apoptosis and migration of human osteosarcoma 143B cells

作　　者：姚娟[1] 翁亚光[1] 严树涓[1] 侯梦一 施琼[1] 左国伟[1] 

机构地区：[1]重庆医科大学检验医学院,临床检验诊断学教育部重点实验室,重庆400016

出　　处：《肿瘤》2015年第2期119-128,共10页Tumor

基　　金：国家自然科学基金资助项目(编号:81102035)~~

摘　　要：目的:探讨肾母细胞瘤过表达(nephroblastoma overexpressed,NOV)基因对人骨肉瘤细胞143B增殖、凋亡和迁移的影响及其可能的作用机制。方法:分别采用半定量反转录聚合酶链反应(reverse transcriptionpolymerase chain reaction,RT-PCR)和蛋白质印迹法筛选NOV低表达的骨肉瘤细胞株。将重组腺病毒Ad NOV和Ad GFP(空载体阴性对照组)分别感染骨肉瘤143B细胞,培养24 h后,在荧光显微镜下观察重组腺病毒Ad NOV和Ad GFP在143B细胞中的感染效率,并通过半定量RT-PCR和蛋白质印迹法检测NOV m RNA和蛋白在143B细胞中的表达情况。分别采用MTT法、FCM法和Transwell小室法检测NOV过表达对143B细胞增殖、凋亡及迁移的影响;半定量RT-PCR法及蛋白质印迹法检测NOV过表达的143B细胞中NOV、抗凋亡因子B细胞淋巴瘤基因-2(B cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)表达水平的变化以及磷酸化p38(phospho-p38,p-p38)、总p38、磷酸化c-Jun氨基末端激酶(phospho-c-Jun N-terminal kinase,p-JNK)和总JNK表达水平的变化。结果:NOV在骨肉瘤细胞143B中的表达水平最低(P<0.05)。荧光显微镜下观察发现,重组腺病毒Ad NOV在143B细胞中的感染效率约为60%;NOV在143B细胞中成功过表达(P<0.05)。NOV过表达可抑制143B细胞的增殖(P<0.05),将细胞周期阻滞于G0/G1期(P<0.05),促进了143B细胞的凋亡(P<0.001);并可提高143B细胞的迁移能力(P<0.001)。NOV过表达后,143B细胞中促凋亡因子Bax表达上调(P<0.001),抑制凋亡因子Bcl-2表达下降(P<0.05);同时,p-p38与p-JNK表达水平均显著上调(P<0.001)。结论:腺病毒介导的NOV过表达抑制了骨肉瘤143B细胞的增殖,促进了其凋亡和迁移,其作用机制可能与p38/丝裂原活化蛋白激酶(mitogenactivated protein kinase,MAPK)和JNK/MAPK信号通路的激活有关。Objective： To investigate the effects of nephroblastoma overexpressed （NOV） gene on proliferation, apoptosis and migration of human osteosarcoma 143B cells in vitro, and to explore its underlying mechanism. Methods： The osteosarcoma cell line with low expression of NOV was screened out by semi- quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. The 143B cells were infected with recombinant adenoviruses AdNOV and AdGFP （as a negative control） and cultured for 24 h. Then the infection efficiencies of recombinant adenoviruses AdNOV and AdGFP in 143B cells were observed under a fluorescence microscope. The expression levels of NOV mRNA and protein were detected by semi-quantitative RT-PCR and Western blotting, respectively. The effects of NOV overexpression on proliferation, apoptosis and migration of 143B cells were measured by MTT, flow cytometry （FCM） and Transwell migration test, respectively. The expression levels of NOV, B cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax） in 143B cells were examined by semi-quantitative RT-PCR and Western blotting. The expression levels of phospho-p38 （p-p38）, total p38, phospho-c- Jun N-terminal kinase （p-JNK） and total JNK were detected by Western blotting. Results： The expression level of NOV was lowest in 143B cells （P 〈 0.05）. The infection efficiency of recombinant adenovirus AdNOV in 143B cells was about 60% under the fluorescence microscope. NOV was successfully overexpressed in 143B cells （P 〈 0.05）. NOV overexpression could inhibit the proliferation of 143B cells （P 〈 0.05）, increase the apoptosis rate （P 〈 0.001） and migration abilitiy （P 〈 0.001） of 143B cells, and arrest the cell cycle at G0/G1 phase （P 〈 0.05）. As compared with the control group, NOV overexpression increased the expession level of Bax （P 〈 0.001） and down-regulated the expression level of Bcl-2 （P 〈 0.05） in 143B cells. At the same time, the expression levels of

关 键 词：骨肉瘤 基因 肾母细胞瘤 细胞增殖 细胞凋亡 肿瘤转移 

分 类 号：R738.1[医药卫生—肿瘤]