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本研究探讨烟酰胺磷酸核糖转移酶(NAMPT)对K562细胞(人慢性髓系白血病细胞株)对伊马替尼敏感性的影响及其相关机制。在体外合成针对NAMPT基因的小干扰RNA(siRNA),转染K562细胞株。将转染后的K562细胞经1μmol/L伊马替尼作用48 h后,采用PI/Calcein染色方法测定细胞存活率;经不同浓度伊马替尼作用48 h后,采用MTS法测定细胞增殖活性,计算半数抑制浓度(IC50)。未经转染的K562细胞经1μmol/L伊马替尼作用3-48 h后,采用Western blot方法检测NAMPT表达的变化。通过对NCBI GEO中基因表达数据的挖掘分析及Western blot方法检测,探讨NAMPT-siRNA和伊马替尼对凋亡相关基因表达的影响。采用荧光素酶报告基因技术,研究NAMPT-siRNA、伊马替尼对NF-κB转录活性的影响。结果表明,1μmol/L伊马替尼作用于K562细胞48 h对NAMPT表达无明显影响。NAMPT-siRNA能够明显抑制K562细胞NAMPT的表达,再经1μmol/L伊马替尼作用后,NAMPT-siRNA干扰组细胞存活率明显降低(P<0.05),提示抑制NAMPT表达能增强K562细胞对伊马替尼的敏感性。不同浓度伊马替尼作用后,干扰组IC50值明显降低(P<0.05);拟合增殖曲线在0.01-0.1μmol/L伊马替尼浓度范围内斜率增大,提示在此范围内NAMPT-siRNA与伊马替尼的协同作用最强。基因表达谱分析及Western blot验证结果都显示,NAMPT-siRNA和伊马替尼处理均可下调NF-κB下游因子抗凋亡基因Bcl-2表达水平,两者同时作用时效果更明显。NAMPT-siRNA及伊马替尼均能降低NF-κB转录活性,两者同时作用效果更显著。结论:伊马替尼可能并非通过调节NAMPT表达影响细胞存活;抑制NAMPT表达能增强K562细胞对伊马替尼的敏感性,可能与抑制NF-κB及其下游因子Bcl-2有关。
This study was to investigate the effects of nicotinamide phosphoribosyltransferase (NAMPT) on the sensitivity of imatinib to K562 cells (human chronic myelogenous leukemia cell line) and its related mechanisms. Small interfering RNA (siRNA) against NAMPT gene was synthesized in vitro and transfected into K562 cell line. The transfected K562 cells were treated with 1 μmol / L imatinib for 48 h, and the cell viability was determined by PI / Calcein staining. After 48 h of imatinib treatment, the cell proliferation activity was determined by MTS assay , Calculate the half inhibitory concentration (IC50). The untransfected K562 cells were treated with 1 μmol / L imatinib for 3-48 h, and the changes of NAMPT expression were detected by Western blot. The effects of NAMPT-siRNA and imatinib on the expression of apoptosis-related genes were investigated by mining the gene expression data in NCBI GEO and Western blotting. Using luciferase reporter gene technology to study NAMPT-siRNA, imatinib on NF-κB transcriptional activity. The results showed that 1μmol / L imatinib on K562 cells for 48 h had no significant effect on NAMPT expression. NAMPT-siRNA could significantly inhibit the expression of NAMPT in K562 cells. After treated with 1μmol / L imatinib, the survival rate of NAMPT-siRNA interference group was significantly decreased (P <0.05), suggesting that inhibition of NAMPT expression could enhance the expression of NAMPT in K562 cells. The sensitivity of The IC50 values of the interference group were significantly decreased (P <0.05) after imatinib treatment at different concentrations. The slope of the fitted proliferation curve increased within the range of 0.01-0.1 μmol / L imatinib, suggesting that the NAMPT- The synergy between siRNA and imatinib is the strongest. The results of gene expression profiling and Western blot showed that both NAMPT-siRNA and imatinib could down-regulate the expression of Bcl-2, an anti-apoptotic gene downstream of NF-κB. Both NAMPT-siRNA and imatinib could reduce the NF-κB transcriptional activity, and the effect of both was more significant. CONCLUSION: Imatinib may not affect cell survival by regulating NAMPT expression. Suppression of NAMPT expression may enhance the sensitivity of K562 cells to imatinib, which may be related to the inhibition of NF-κB and its downstream factor Bcl-2.