NACK的下调对急性T淋巴细胞白血病Jurkat细胞增殖、凋亡的影响

Effect of NACK knockdown on the proliferation and apoptosis of T-cell acute lymphoblastic leukemia Jurkat cells

目的探讨NACK基因表达下调对急性T淋巴细胞白血病Jurkat细胞增殖、凋亡的影响及作用机制。方法通过慢病毒转染技术感染Jurkat细胞,使NACK基因表达下调,应用实时荧光定量PCR(qRT-PCR)、Western blot检测NACK基因的沉默效率;CCK-8法、流式细胞术检测NACK下调后对Jurkat细胞增殖、凋亡的影响;Western blot检测Notch1信号传导通路下游相关蛋白Hes1、c-Myc的表达情况。结果NACK-短发夹RNA(shRNA)通过慢病毒载体成功转染Jurkat细胞后,NACK mRNA及蛋白表达量明显降低(P<0.05);与阴性对照组和空白对照组比较,CCK-8法显示实验组细胞增殖受到明显抑制[实验组、阴性对照组和空白对照组细胞抑制率分别为(37.27±4.48)%、(4.25±2.10)%和(2.43±1.40)%](F=132.640,P<0.05),流式细胞术检测实验组细胞凋亡明显增加[实验组、阴性对照组和空白对照组细胞凋亡率分别为(26.38±3.03)%、(6.07±2.61)%和(3.40±1.98)%](F=90.534,P<0.05);Western blot结果证实Notch1通路相关蛋白Hes1、c-Myc的表达量较阴性对照组及空白对照组下调,差异有统计学意义(P<0.05)。结论靶向沉默NACK可下调Notch1信号通路相关蛋白的表达,导致Jurkat细胞增殖抑制、细胞凋亡增多,从而发挥其抗T淋巴细胞白血病的作用。

Objective To investigate the effect and mechanism of NACK knockdown on the proliferation and apoptosis of T-cell acute lymphoblastic leukemia(T-ALL)Jurkat cells.Methods Lentivirus transfection technology was used to transfect Jurkat cells and knock down NACK gene.Real time fluorescent quantitative PCR and Western blot were used to detect the silencing efficiency of NACK gene.CCK-8 method and flow cytometry were used to detect the effects of NACK knockdown on the proliferation and apoptosis of Jurkat cells.The expressions of protein related with Notch1 pathway,such as Hes1 and c-Myc,were detected by Western blot.Results After NACK-shRNA was successfully transfected into Jurkat cells by lentiviral vector,the expression of NACK mRNA and protein was reduced signi-ficantly(P<0.05).Compared with the negative control group and the blank control group,the CCK-8 method showed that the cell proliferation in the experimental group was significantly inhibited[The inhibition rates of cell proliferation in the experimental group,negative control group and blank control group were(37.27±4.48)%,(4.25±2.10)%and(2.43±1.40)%,respectively](F=132.640,P<0.05),and the flow cytometry test showed that the apoptosis in the experimental group increased significantly[The apoptosis rates of experimental group,negative control group and blank control group were(26.38±3.03)%,(6.07±2.61)%and(3.40±1.98)%,respectively](F=90.534,P<0.05).Western blot results confirmed that the expression of Notch1 pathway-related proteins Hes1 and c-Myc was down-regulated compared with the negative control group and the blank control group,and the difference was statistically significant(P<0.05).Conclusions Targeting silent NACK can down-regulate the expression of Notch1 pathway-related proteins,which leads to the inhibition of Jurkat cell proliferation and increased apoptosis,thereby exerting its anti-T-ALL effect.