shRNA干扰沉默Mcl-1基因对淋巴瘤细胞系增殖的影响

目的研究慢病毒介导的髓细胞白血病基因(Mcl-1)短发夹状RNA(shRNA)干扰对淋巴瘤细胞系SNK-6增殖和凋亡的影响。方法设计以Mcl-1为靶点的shRNA并构建携带此shRNA的慢病毒载体,转染淋巴瘤细胞系SNK-6,荧光定量PCR(qPCR)及Western blot方法检测病毒感染前后Mcl-1 mRNA及蛋白表达变化,四甲基偶氮唑盐法(MTT法)和流式细胞仪分析细胞增殖和凋亡变化的情况。结果成功构建Mcl-1-shRNA慢病毒表达载体,并可有效感染淋巴瘤细胞株SNK-6,感染后SNK-6细胞Mcl-1基因的mRNA水平及蛋白水平表达明显下调,MTT法检测显示慢病毒介导的Mcl-1基因shRNA干扰与对照病毒组相比可明显抑制SNK-6细胞增殖(31.6±3.3)%vs(5.8±2.7)%,P<0.01],流式细胞仪测定感染后细胞凋亡明显高于对照病毒组(28.9±2.1)%vs(5.3±1.5)%,P<0.01]。结论慢病毒介导的Mcl-1基因shRNA干扰技术可特异性阻断SNK-6细胞Mcl-1基因的表达,抑制细胞的增殖并促进细胞的凋亡。

Effect of shRNA interference targeting myeloid leukemia-1 gene on proliferation in lymphoma cell line

Objective To explore the effect of lentivirus-mediated short hairpin RNA(shRNA) interference targeting myeloid leukemia-1(Mcl-1) gene on the proliferation and apoptosis of lymphoma cell line SNK-6.Methods The shRNA targeting Mcl-1 gene was designed and the lentiviral vector carrying the shRNA was constructed.Lymphoma SNK-6 cells were transfected with the recombinant lentiviral vectors(shRNA group) and the blank lentiviral vectors(control group),respectively.Real-time PCR and Western blotting were used to analyze the mRNA and protein expression of Mcl-1 gene in SNK-6 cells before and after transfection.MTT assay and flow cytometry were applied to detect the proliferation and apoptosis of the transfected SNK-6 cells,respectively.Results The shRNA targeting Mcl-1 gene was successfully inserted into the lentiviral vectors,which could effectively infect lymphoma SNK-6 cells.The mRNA and protein expression of Mcl-1 gene in the shRNA group significantly decreased as compared with those in the control group.The MTT assay results suggested that the proliferation of SNK-6 cells was significantly inhibited in the shRNA group as compared with that in the control group (31.6±3.3)% vs(5.8±2.7)%,P<0.01].The flow cytometry showed that the apoptosis rate of the shRNA group was significantly higher than that of the control group (28.9±2.1)% vs(5.3±1.5)%,P<0.01].Conclusion Lentivirus-mediated shRNA interference targeting Mcl-1 gene can specifically block Mcl-1 gene expression,and inhibit the proliferation and promote the apoptosis of SNK-6 cells.