APE1基因沉默增强骨肉瘤U2-OS细胞硼替佐米治疗敏感性的实验研究  

APE1 Gene Silencing Promotes the Sensitivity of Osteosarcoma U2-OS Cells to Bortezomib

作　　者：多健[1] 王国文[1] 韩秀鑫[1] 杨吉龙[1] 孙建合[2] 

机构地区：[1]天津医科大学附属肿瘤医院骨和软组织肿瘤科,天津市肿瘤防治重点实验室,天津市300060 [2]天津医科大学附属肿瘤医院麻醉科,天津市300060

出　　处：《中国肿瘤临床》2012年第8期429-432,共4页Chinese Journal of Clinical Oncology

基　　金：天津市自然科学基金(编号:08JCYBJC05500)资助~~

摘　　要：目的:探讨脱嘌呤/脱嘧啶核酸内切酶1(APE1)基因沉默对蛋白酶体抑制剂硼替佐米(bortezomib,PS-341)抑制骨肉瘤U2-OS细胞增殖作用的影响及其生物学机制方法:将APE1特异性shRNA的重组质粒,稳定转染人骨肉瘤U2-OS细胞,采用聚合酶链反应和免疫印迹法检测转染前后U2-OS细胞中APE1的表达,采用四甲基偶氮唑盐法观察PS-341和APE1-siRNA对人骨肉瘤U2-OS细胞生长的抑制作用,采用免疫印迹法检测PS-341和APE1-siRNA对U2-OS细胞中APE1和胞核NF-λB的表达的影响。结果:细胞稳定转染APE1-siRNA重组质粒后,APE1mRNA和蛋白表达分别下降约46.1%和62.6%,MTT法检测U2-OS细胞增殖受到抑制。转染前后U2-OS细胞PS-341的IC50值分别为371.54 nmoL/L与109.64 nmoL/L,两者比较差异有统计学意义(P<0.01)。Western blot结果显示PS-341和APE1-siRNA均抑制U2-OS细胞胞核中NF-λB的表达,两者联合应用抑制效果更明显。结论:APE1-shRNA质粒转染骨肉瘤U2-OS细胞后,肿瘤细胞的增殖率降低,对PS-341抑制U2-OS细胞的增殖具有协同作用。Objective： To investigate the effects of apurinic / apyrimidinic endonuclease 1 （ APE1 ） on the inhibitory action of bortezomib on human osteosarcoma U2-OS cells and the underlying biological mechanisms. Methods： An shRNA plasmid that targets APE 1 was constructed and transfected into U2-OS cells. The mRNA and protein levels of APE 1 were detected via reverse transcription polymerase chain reaction and Western blot analysis. The inhibition of cell proliferation induced by PS-341 and APE 1-siRNA was examined with an 3- （ 4, 5-dimethylthiazol-2-yl ） 2, 5-diphenyl tetrazolium bromide assay. The change in nuclear NF-κB and APE1 expression induced by PS-341 and APE1 in osteosarcoma U2-OS cells was examined using Western blot analysis. Results： The APEI-shRNA expression plasmid was successfully constructed and transfected into U2-OS cells. The expression inhibition rate was about 47.6 % at the mRNA level, and was about 62.6 % at the protein level. Osteosarcoma cell proliferation was inhibited, as indicated by the MTT analysis. The median inhibitory concentration of PS-341 was 371.54 nmoL/L before APEI-shRNA transfection, which significantly decreased to 109.64 nmoL/L after APE 1-shRNA transfection （ P 〈 0.01 ）. The Western blot analysis indicated that both PS-341 and APE 1-siRNA downregulated nuclear NF-κB protein expression in the U2-OS ceils. The effect was more significant than that of combination of the above two. Conclusion： After APEI-shRNA plasrnid transfection into the osteosarcoma U2-OS cells, APE1 expression was inhibited at the protein and mRNA levels. The osteosarcoma cell proliferation rate was also decreased, and the PS-341 inhibitory effect on the osteosarcoma cells was promoted. The biological mechanisms may be related to the downregulation of nuclear NF-κB expression.

关 键 词：骨肉瘤 蛋白酶体抑制剂 脱嘌呤脱嘧啶核酸内切酶1 NF-ΚB蛋白 

分 类 号：R738.1[医药卫生—肿瘤]