摘要
目的探讨小干扰RNA(siRNA)沉默烟酰胺磷酸核糖转移酶(NAMPT)基因表达对人多发性骨髓瘤U266细胞增殖、凋亡的影响及其相关机制。方法在体外合成针对NAMPT基因的siRNA并转染U266细胞,实验分为si-NAMPT组(转染siRNA-NAMPT的U266细胞)、si-NC组(转染阴性对照siRNA的U266细胞),采用四甲基偶氮唑盐(MTT)法检测转染后U266细胞增殖情况,流式细胞术检测细胞凋亡情况,蛋白质印迹法检测转染后各组细胞NAMPT、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、糖原合成酶激酶3β(GSK-3β)、磷酸化GSK-3β(p-GSK-3β)、β-catenin表达水平。结果si-NAMPT组转染48、72 h后与si-NC组比较,对U266细胞增殖抑制作用(570 nm处吸光度值)增加(48 h:0.78±0.06比1.62±0.11;72 h:1.23±0.14比2.37±0.18),差异均有统计学意义(t=3.54,P=0.034;t=4.72,P<0.01)。si-NAMPT组与si-NC组相比,细胞早期凋亡率升高[(53.42±0.25)%比(25.98±3.18)%],差异有统计学意义(t=4.41,P<0.01)。与si-NC组相比,si-NAMPT组p-AKT、p-GSK-3β、NAMPT、β-catenin蛋白水平均降低(均P<0.05)。结论沉默NAMPT基因可明显抑制U266细胞增殖并诱导细胞凋亡,其可能通过抑制AKT-GSK-3β-β-catenin信号通路发挥作用。
Objective To investigate the effect of small interfering RNA(siRNA)silencing nicotinamide phosphoribosyltransferase(NAMPT)gene expression on proliferation and apoptosis of human multiple myeloma U266 cells and its mechanism.Methods In vitro,NAMPT gene-specific siRNA was synthesized to transfect U266 cells.The experiment was divided into si-NAMPT group(transfected siRNA-NAMPT U266 cells)and si-NC group(transfected negative control siRNA U266 cells).The proliferation of U266 cells after transfection was detected by the methyl thiazolyl tetrazolium(MTT)method,and the apoptosis was detected by flow cytometry.Western blot was used to detect the expression levels of NAMPT,protein kinase B(AKT),phosphorylation-AKT(p-AKT),glycogen synthase kinase-3β(GSK-3β),phosphorylation-GSK-3β(p-GSK-3β),andβ-catenin proteins in each group after transfection.Results Compared with the si-NC group,the proliferation inhibition of U266 cells(absorbance value at 570 nm)increased after transfection for 48 h and 72 h in the si-NAMPT group(48 h:0.78±0.06 vs.1.62±0.11;72 h:1.23±0.14 vs.2.37±0.18),and the differences were statistically significant(t=3.54,P=0.034;t=4.72,P<0.01).The early apoptotic rate of cells in the si-NAMPT group increased compared with si-NC group[(53.42±0.25)%vs.(25.98±3.18)%],and the difference was statistically significant(t=4.41,P<0.01).Compared with the si-NC group,the levels of p-AKT,p-GSK-3β,NAMPT andβ-catenin proteins were significantly reduced in the si-NAMPT group(all P<0.05).Conclusion Silence of NAMPT gene can significantly inhibit U266 cell proliferation and induce cell apoptosis,and it may play a role by inhibiting the AKT-GSK-3β-β-catenin signaling pathway.
作者
王亚茹
马艳萍
Wang Yaru;Ma Yanping(Department of Hematology,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China)
出处
《白血病．淋巴瘤》
CAS
2021年第1期27-30,共4页
Journal of Leukemia & Lymphoma
基金
山西省重点研发计划(201903D321099)
山西医科大学大学生创新创业校级项目(20172108)。
