Objective: To investigate the effects of apurinic / apyrimidinie endomiclease 1 ( APE1 ) on the inhibitory action of bortezomib on human osteosarcoma U2-OS cells and the underlying biological mechanisms. Methods: An shRNAplasmid that targets APE 1 was constructed and transfected into U2-OS cells. The mRNA and protein levels of APE1 were detected via reverse transcription polymerase chain reaction and Western blot analysis. The inhibition of cell proliferation induced by PS-341 and APEl-siRNA was examined with an 3- ('I. 5-dimethy3thiazo3-2-yl) 2, 5-diphenyl tetrazolium bromide assay. The change in nuclear NF-kB and APE 1 expression induced by PS-341 andAPEl in osteosarcoma (J2-OS cells was examined using Western blot analysis. Results: The APEl-shRNA expression plasmid was successfully constructed and transfected into U2-OS cells. The expression inhibition rate was about '17.6 % 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 APEl-shRNA trans lection, which significantly decreased to 109.64 nrnoL/L after APE1 -shRNA transfection ( P < 0.01 ). The Western blot analysis indicated that both PS-341 and APE 1 -siRN A downregulated nuclear NF-k B protein expression in the U2-QS cells. The effect was more significant than that of combination of the above two. Conclusion: After APEl-shRNA plasmid 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 downregulaiion of nuclear NF-kB expression.%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-κB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-κB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.推测其生物学机制可能与下调胞核 NF-κB蛋白表达有关.
展开▼
