miRNA-145对白血病HuT 78细胞凋亡的影响及其机制研究

Effect of MiRNA-145 on Apoptosis of Leukemia HuT 78 Cells and Its Mechanism

目的:探讨miRNA-145对白血病细胞凋亡的影响及其机制。方法:利用Lipofectamine 2000脂质体将miRNA-145模拟物(mimic)及阴性对照转染至白血病细胞后,采用MTT实验检测miR-145对细胞增殖能力的影响,采用流式细胞术检测miR-145对白血病细胞的细胞周期及凋亡影响,采用Western blot实验检测BCL-2、CDK6、Cyclin D1、BAX、PI3K、p-PI3K、p-AKT及AKT表达水平。结果:miR-145模拟物转染HuT 78细胞后,miR-145模拟物相对水平为2.3±0.2,显著高于空白对照组及miR-NC组(P<0.05)。miR-145模拟物转染至HuT78细胞后,HuT 78细胞增殖水平明显低于空白对照及miR-NC组(P<0.05)。转染miRNA-145模拟物后可促使HuT 78细胞的G0/G1期细胞数量增多,S期及G2/M期细胞数量显著减少(P<0.05)。AnnexinV/PI双染流式细胞术实验发现,转染miRNA-145模拟物后HuT 78细胞凋亡率为17.6%±3.4%,显著高于对照组及miRNA-NC组(P<0.05)。miR-145模拟物可显著降低HuT 78细胞内BCL-2、CDK6、Cyclin D1的表达水平(P<0.05);miR-145模拟物可使HuT 78细胞内BAX表达水平增高(P<0.05)。HuT 78细胞转染miR-145模拟物后,HuT78细胞的PI3K、p-PI3K、AKT及p-AKT的表达显著低于空白对照及miR-NC组(P<0.05)。结论:上调miR-145可抑制白血病细胞的增殖能力,并促进细胞发生凋亡,其机制可能与PI3K/AKT信号通路受到抑制有关。

Objective:To investigate the effect and mechanism of miRNA-145 on leukemic cell apoptosis.Methods:After transfection of miRNA-145 mimic and negative control mimic in leukemia cells by Lipofectamine 2000 liposome,the MTT assay was used to detect the effect of miRNA-145 on cell proliferation.Flow cytometry was used to detect the effect of miRNA-145 on cell cycle and apoptosis.Western blotting assay was used to detect the expression levels of BCL-2,CDK6,Cyclin D1,BAX,PI3 K p-PI3 K,p-AKT and AKT.Results:The relative level of microRNA in HuT 78 cells transfected with miRNA-145 was 2.3±02,which was significantly higher than that in blank control group and miRNANC group(P<0.05).MTT assay showed that the proliferation level of HuT 78 cells transfected with miRNA-145 mimic was significantly lower than that of blank control and miRNA-NC group(P<0.05).Flow cytometry showed that the cells at G0/G1,S and G2/M phase of HuT 78 cells were significantly decreased after transfection with miRNA-145 mimic(P<0.05).Annexin V/PI double staining assay showed that the apoptosis rate of HuT 78 cells was 17.6%±3.4%,which was significantly higher than that in blank control group and miRNA-NC group(P<0.05).Western blot showed that the expression levels of BCL-2,CDK6 and Cyclin D1 in HuT 78 cells were significantly lower than those in blank control and miRNA-NC group(P<0.05),and BAX expression in HuT 78 cells was significantly higher than that in blank control and miRNA-NC group(P<0.05).Western blot showed that expression of PI3 K,p-PI3 K,AKT and p-AKT in HuT 78 cells transfected with miRNA-145 mimic were significantly lower than that in blank control and miRNA-NC group(P<0.05).Conclusion:Upregulation of miRNA-145 may inhibit the proliferation of leukemia cells and promote the apoptosis,which may be related with the inhibition of PI3 K/AKT signaling pathway.