摘要
目的:探讨miR-30a的高表达对慢性髓系白血病K562细胞株的促凋亡作用及其机制。方法:构建pEGFP-pre-miR-30a重组质粒并转染K562细胞,采用实时定量PCR法检测miR-30a和BCR/ABL mRNA的表达水平,应用annexinV-FITC/PI双集流式细胞术(flow cytometry,FCM)分析K562细胞凋亡百分率,应用蛋白印迹法(Western blot)分析BCR/ABL融合蛋白、凋亡相关蛋白BCL-2、BAX及PTEN、AKT、p-AKT的变化。结果:测序与酶切图谱证实成功构建重组质粒。与pEGFP-C1-K562阴性对照组和K562空白对照组比,重组质粒pEGFP-pre-miR-30a转染组miR-30a表达水平显著升高,BCR/ABL mRNA与蛋白表达明显下调,凋亡细胞所占比例明显增加(P<0.05),抗凋亡蛋白BCL-2水平降低,促凋亡蛋白BAX表达增加,抑癌蛋白PTEN表达明显上升,AKT无明显变化,但活性形式的p-AKT显著降低,差异均有统计学意义。结论:高表达的miR-30a能抑制K562细胞癌基因BCR/ABL mRNA及蛋白的表达并促进细胞凋亡,其机制可能与抑制BCR/ABL-PTEN/AKT信号通路的活性有关。
Objective:To investigate the pro-apoptotic effect and mechanism of miR-30a overexpression on chronic myeloid leukemia K562 cells.Methods:The k562 cells were transfected with the recombinant plasmid pEGFP-pre-miR-30a,the real-time quantitative PCR was used to detect the level of miR-30a and BCR/ABL,and then the cell apoptosis was assessed by flow cytometry with AnnexinV-FITC/PI double staining.Western blot was used to detect the expression of BCR/ABL protein,apoptosis-related protein BCL-2 and BAX,PTEN,AKT and p-AKT.Results:Sequencing and digestion map indicated that the recombinant plasmid was constructed successfully.Compared with 2 control groups,the miR-30a expression in k562 cells transfected with recombinant plasmid pEGFP-pre-miR-30a was obviously upregulated.The expression of BCR/ABL mRNA and BCR/ABL protein was both significantly down-regulated.Apoptotic rate was significantly enhanced(both P<0.05),and the expression of anti-apoptotic protein BCL-2 was down-regulated while the expression of pro-apoptotic protein BAX was up-regulated.The level of PTEN was significantly up-regulated in omparison with control groups,no variation was found in total AKT,but the expression of p-AKT was down-regulated.Conclusion:The overexpression of miR-30a is abled to down-regulate the level of BCR/ABL mRNA and BCR/ABL protein,and increase apoptotic rate,its mechanism may be related with inhibition of the activity of BCR/ABL-PTEN/AKT signaling pathway.
作者
徐敏
高雯琬
雒钰杰
王毅
陶崑
XU Min;GAO Wen-Wan;LUO Yu-Jie;WANG Yi;TAO Kun(Department of Immunology,College of Basic Medical Science,Chongqing Medical University,Chongqing 400016,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2019年第2期396-402,共7页
Journal of Experimental Hematology
基金
重庆市渝中区科委课题(20130139)
