miR-431-5p抑制MDM2表达影响白血病细胞增殖、凋亡的机制研究

miR-431-5p Affects Proliferation and Apoptosis of Leukemia Cells by Inhibiting MDM2 Expression

目的探讨miR-431-5p靶向MDM2对白血病细胞增殖、凋亡的影响。方法qRT-PCR检测白血病患者、正常人骨髓细胞和白血病细胞HL-60、K562中miR-431-5p的表达。以K562细胞为研究对象,将其随机分为miR-NC组(转染miR-NC)、miR-431-5p组(转染miR-431-5p)、miR-431-5p+pcDNA3.1组(miR-431-5p和pcDNA3.1共转染)和miR-431-5p+pcDNA3.1-MDM2组(miR-431-5p和pcDNA3.1-MDM2共转染)。MTT法检测细胞增殖活力,流式细胞术检测细胞凋亡,Western blot检测MDM2、CyclinD1、P21、Bax和Bcl-2蛋白表达水平。采用双荧光素酶报告基因法和Western blot验证miR-431-5p和MDM2的靶向关系。结果与正常人骨髓细胞相比,白血病患者骨髓细胞和白血病细胞HL-60、K562中miR-431-5p的表达显著下降,差异有统计学意义(P<0.05)。与miR-NC组相比,miR-431-5p组K562细胞MDM2、Cyclin D1和Bcl-2蛋白的表达明显下调,P21和Bax蛋白的表达明显上调,细胞的增殖活力显著减弱,凋亡率显著升高,差异有统计学意义(P<0.05)。与miR-431-5p+pcDNA3.1组比较,miR-431-5p+pcDNA3.1-MDM2组K562细胞MDM2、Cyclin D1和Bcl-2蛋白的表达明显升高,P21和Bax蛋白的表达明显降低,细胞的增殖活力显著升高,凋亡率显著降低,差异有统计学意义(P<0.05)。结论miR-431-5p通过靶向下调MDM2可抑制白血病细胞增殖,促进白血病细胞凋亡。

Objective To investigate the effects of miR-431-5p on proliferation and apoptosis of leukemia cells by targeting MDM2.Methods The expression of miR-431-5p was detected by qRT-PCR in leukemia patients,normal human bone marrow cells and leukemia HL-60 and K562 cells.K562 cells were randomly divided into miR-NC group(transfection with miR-NC),miR-431-5p group(transfection with miR-431-5p),miR-431-5p+pcDNA3.1 group(co-transfection with miR-431-5p and pcDNA3.1),and miR-431-5p+pcDNA3.1-MDM2 group(co-transfection with miR-431-5p and pcDNA3.1-MDM2).Cell proliferation was determined by MTT assay.The apoptosis was measured by flow cytometry.The protein expression of MDM2,CyclinD1,P21,Bax and Bcl-2 was detected by Western blot.The dual luciferase reporter gene method and Western blot were used to verify the targeting relationship between miR-431-5p and MDM2.Results Compared with normal bone marrow cells,the expression of miR-431-5p significantly decreased in leukemia patients and leukemia HL-60 and K562 cells(P<0.05).Compared with miR-NC group,the expression of MDM2,Cyclin D1 and Bcl-2 and the activity of proliferation decreased and the expression of P21 and Bax and the rate of apoptosis increased in K562 cells in miR-431-5p group(P<0.05).Compared with miR-431-5p+pcDNA3.1 group,the expression of MDM2,Cyclin D1 and Bcl-2 and the activity of proliferation increased and the expression of P21 and Bax and the rate of apoptosis decreased in K562 cells in miR-431-5p+pcDNA3.1-MDM2 group(P<0.05).Conclusion miR-431-5p can inhibit proliferation and promote apoptosis in leukemia cells by targeted down-regulation of MDM2.