下调ANRIL对Kasumi-1细胞增殖和凋亡的影响及其相关作用机制研究

Effect of Down-Regulation of ANRIL on Proliferation and Apoptosis of Kasumi-1 Cells and Its Potential Mechanism

目的:探讨下调ANRIL(Antisense non-coding RNA in the INK4 Locus)表达对Kasumi-1细胞增殖和凋亡的影响及其相关分子机制。方法:使用重组慢病毒构建下调ANRIL表达的Kasumi-1细胞(sh-ANRIL组)及其对照组细胞(sh-NC组),荧光显微镜观察慢病毒对细胞的转染效率,RT-qPCR检测敲低效率及急性髓细胞白血病(AML)患者外周血和骨髓中ANRIL的表达水平。CCK-8法和流式细胞术检测下调ANRIL表达对Kasumi-1细胞增殖和凋亡的影响。Western blot检测下调ANRIL表达后Kasumi-1细胞中PI3K、AKT、p-AKT等相关蛋白分子的表达。结果:ANRIL在AML患者外周血和骨髓中呈高表达;重组慢病毒对Kasumi-1细胞的转染效率>90%,敲低效率为70%;CCK-8结果显示,下调Kasumi-1细胞中ANRIL表达能够显著抑制细胞增殖,当DNR作用24、48、72 h,sh-ANRIL组的细胞增殖抑制率分别为(47.40±1.49)%、(69.11±0.51)%和(91.82±1.10)%,均显著高于sh-NC组(40.69±1.70)%(P<0.05)、(58.93±1.74)%(P<0.01)和(82.93±0.37)%(P<0.001);流式细胞术分析显示,sh-ANRIL组细胞凋亡率为(10.29±0.58)%,显著高于sh-NC组的(5.42±0.67)%(P<0.01),当DNR作用24 h,sh-ANRIL组细胞凋亡率为(54.41±1.69)%,显著高于sh-NC组的(38.28±1.42)%(P<0.001);Western blot结果显示,sh-ANRIL组PI3K、p-AKT、PCNA、BCL-2蛋白水平较sh-NC组显著降低,而BAX蛋白的表达增加。结论:ANRIL可能通过PI3K/AKT信号通路影响Kasumi-1细胞增殖和凋亡,ANRIL是AML的潜在治疗靶点。

Objective:To investigate the down-regulation of ANRIL(Antisense non-coding RNA in the INK4 Locus)effects on proliferation and apoptosis of Kasumi-1 cells and its related molecular mechanism.Methods:Recombinant lentivirus was used to construct ANRIL down-regulation Kasumi-1 cells(sh-ANRIL group)and its control cells(sh-NC group).A fluorescence microscope was used to observe the transfection efficiency,RT-qPCR was used to detect knockdown efficiency and ANRIL expression in PBMCs and MBMCs of patients with acute myeloid leukemia(AML).Proliferation and apoptosis of Kasumi-1 cells were assayed by CCK-8 method and flow cytometry.Western blot was employed to detect the expression of PI3K,AKT,p-AKT,and relevant protein after down-regulation of ANRIL in Kasumi-1 cells.Results:ANRIL overexpressed significantly in PBMCs and MBMCs of patients with AML,the transfection efficiency of recombinant lentivirus carrying sh-ANRIL and sh-NC on Kasumi-1 cells exceeded 90%,and the knockdown efficiency was 70%.When DNR was administrated for 24,48,and 72 hours,the cell inhibition rate of the sh-ANRIL group was(47.40±1.49)%,(69.11±0.51)%and(91.82±1.10)%,which were significantly higher than those of the sh-NC group,respectively(P<0.05).The apoptotic rate in the sh-ANRIL group was(10.29±0.58)%,which was significantly higher than(5.42±0.67)%of the sh-NC group(P<0.01).After DNR treatment for 24 hours,the apoptotic rate of the sh-ANRIL group was(54.41±1.69)%,which was significantly higher than(38.28±1.42)%of sh-NC group(P<0.001).Western blot revealed that the protein levels of PI3K,p-AKT,PCNA,and BCL-2 in the sh-ANRIL group were reduced significantly than those in the sh-NC group,while the BAX protein expression increased.Conclusion:ANRIL may affect the proliferation and apoptosis of Kasumi-1 cells through PI3K/AKT signaling pathway.ANRIL is a potential therapeutic target for AML.