miR-181a-5p和BACH2表达对白血病CCRF-CEM细胞凋亡和侵袭的影响

Effects of miR-181a-5p and BACH2 expressions on apoptosis and invasion of leukemic CCRF-CEM cells

目的:探讨微小RNA-181a-5p(miR-181a-5p)靶向BTB与CNC同源基因2 (BACH2)对急性淋巴细胞白血病(ALL)细胞凋亡和侵袭的调控作用,并阐明其作用机制。方法:体外构建BACH2过表达重组质粒(overExpBACH2)、miR-181a-5p inhibitor和inhibitor-NC,转染人ALL T淋巴细胞CCRF-CEM,实时荧光定量PCR (RT-qPCR)法和免疫荧光染色检测转染效果。将CCRF-CEM细胞分为对照组、 inhibitor-NC组、 miR-181a-5pinhibitor组、 BACH2过表达(overExpBACH2)组、 miR-181a-5pinhibitor+空载体(EV)组和miR-181a-5pinhibitor+overExpBACH2组。CCK-8法检测各组细胞增殖活性,流式细胞术检测G_(2)期细胞百分率和细胞凋亡率。Western blotting法检测各组细胞中细胞周期蛋白D3 (CyclinD3)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)和含半胱氨酸的天冬氨酸蛋白水解酶3 (Caspase-3)蛋白表达水平,Transwell小室实验检测各组细胞中侵袭细胞数,RT-qPCR法检测各组细胞中基质金属蛋白酶9 (MMP-9)、基质金属蛋白酶14 (MMP-14)和BACH2 mRNA及miR-181a-5p表达水平。结果:miR-181a-5p inhibitor和overExpBACH2质粒均成功转染CCRF-CEM细胞。与对照组比较,miR-181a-5pinhibitor组、overExpBACH2组、miR-181a-5p inhibitor+EV组和miR-181a-5p inhibitor+overExpBACH2组细胞增殖活性明显降低(P<0.05),G_(2)期细胞百分率和细胞凋亡率明显升高(P<0.05),细胞中CyclinD3蛋白表达水平明显降低(P<0.05),Caspase-3蛋白表达水平和Bax/Bcl-2比值明显升高(P<0.05),侵袭细胞数明显减少(P<0.05),细胞中MMP-9和MMP-14 mRNA及miR-181a-5p表达水平明显降低(P<0.05),BACH2mRNA表达水平明显升高(P<0.05)。与miR-181a-5p inhibitor组和overExpBACH2组比较,miR-181a-5p inhibitor+overExpBACH2组细胞中上述指标变化趋势更明显。结论:miR-181a-5p可靶向BACH2进而调控ALL细胞侵袭和凋亡,为临床靶向治疗ALL提供了新的靶点。

Objective:To discuss the regulatory effect of microRNA-181a-5p(miR-181a-5p)targeting BTB and CNC homology 2(BACH2)on the apoptosis and invasion of the acute lymphoblastic leukemia(ALL)cells,and to clarify its mechanism.Methods:The BACH2 over-expression recombinant plasmid(overExpBACH2),miR-181a-5p inhibitor,and inhibitor-NC were constructed in vitro,and the transfection effect after transfected with human ALL T lymphocytes CCRF-CEM was detected by real-time fluorescence quantitative PCR(RT-qPCR)and immunofluorescence staining.The CCRF-CEM cells were divided into control group,inhibitor-NC group,miR-181a-5p inhibitor group,BACH2 over-expression(overExpBACH2)group,miR-181a-5p inhibitor+empty vector(EV)group,and miR-181a-5p inhibitor+overExpBACH2 group.The proliferation activities of cells in various groups were detected by CCK-8 assay;the percentages of cells at G_(2) phase and apoptotic rates of cells in various groups were detected by flow cytometry;the expression levels of CyclinD3,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cysteinyl aspartate specific proteinase-3(Caspase-3)proteins in cells in various groups were detected by Western blotting method;the numbers of invasion cells in various groups were detected by Transwell chamber test;the expression levels of matrix metalloproteinase-9(MMP-9),matrix metalloproteinase 14(MMP-14),BACH2 mRNA,and miR-181a-5p in cells in various groups were detectd by RT-qPCR method.Results:Both miR-181a-5p inhibitor and overExpBACH2 vector were successfully transfected into the CCRF-CEM cells.Compared with control group,the proliferation activities of the cells in overExpBACH2 group and miR-181a-5p inhibitor+EV group were decreased(P<0.05),and the percentages of the cells at G_(2) phase and the apoptotic rates were increased(P<0.05);the expression levels of CyclinD3 protein in the cells were decreased(P<0.05),the expression levels of Caspase-3 protein and the ratios of Bax/Bcl-2 were increased(P<0.05),the numbers of invasion cells were decreased(P<0.05),the expression levels of MMP-9,MMP-14 mRNA,and miR-181a-5p were decreased(P<0.05),and the BACH2 mRNA expression levels were increased(P<0.05).Compared with miR-181a-5p inhibitor group and overExpBACH2 group,the change trends of the above indexes in miR-181a-5p inhibitor+overExpBACH2 group were more obvious.Conclusion:miR-181a-5p can target BACH2 and regulate the apoptosis and invasion of the ALL cells,which provides the new ideas for the clinical target therapy of ALL.