miR-424靶向MEK1对白血病HL-60细胞系增殖、凋亡及阿霉素耐药性的影响

Effects of MEK1 targeting miR-424 on proliferation,apoptosis,and adriamycin resistance of leukemia HL-60 cell line

目的探究miR-424对白血病HL-60细胞增殖、凋亡及阿霉素(ADM)耐药性的影响及其可能的作用机制。方法体外培养正常人的外周血单核细胞(PBMC)、人原髓细胞白血病细胞(HL-60),向细胞HL-60转染miR-424阴性对照、miR-424 mimic,将细胞分为空白对照组、NC组和miR-424 mimic组,以PBMC为正常对照组。实时荧光定量PCR(qRT-PCR)检测PBMC及各组HL-60细胞中miR-424、丝裂原活化蛋白激酶激酶1(MEK1)mRNA相对表达水平。CCK8法检测各组HL-60细胞增殖情况。流式细胞术检测各组HL-60细胞凋亡情况。对各组HL-60细胞进行ADM处理,CCK8法检测各组HL-60细胞对ADM敏感性,双荧光素酶报告基因检测miR-424与MEK1的靶向关系,蛋白印迹法(WB)检测各组HL-60细胞MEK1、Ki67、Bcl-2、Bax蛋白相对表达水平。结果与正常对照组相比,空白对照组HL-60细胞中miR-424相对表达水平显著降低,MEK1 mRNA相对表达水平显著升高,差异均有统计学意义(P<0.05);与空白对照组、NC组相比,miR-424 mimic组HL-60细胞增殖率、ADM IC50、MEK1 mRNA、Ki67、Bcl-2、MEK1蛋白相对表达水平显著降低,miR-424、细胞凋亡率、Bax蛋白相对表达水平显著升高,差异均有统计学意义(P<0.05);miR-424 mimic+WT-MEK13'-UTR组的荧光素酶相对活性低于miR-424 NC+WT-MEK13'-UTR组,差异有统计学意义(P<0.05)。结论miR-424在白血病HL-60细胞中低表达,miR-424过表达可能通过靶向抑制MEK1表达,抑制白血病HL-60细胞增殖、促进凋亡,提高细胞对阿霉素的敏感性。

Objective To investigate the effects of miR-424 on the proliferation,apoptosis and adriamycin(ADM)resistance of leukemia HL-60 cells and its possible mechanism.Methods Normal human peripheral blood monocytes(PBMC)and human myeloid leukemia cells HL-60 were cultured in vitro,and HL-60 cells were transfected with miR-424 negative control and miR-424 mimic.The cells were divided into blank control group,NC group,and miR-424 mimic group,and PBMC were used as normal control group.The relative expression levels of miR-424,mitogen-activated protein kinase kinase 1(MEK1)mRNA in PBMC and HL-60 cells were detected by qRT-PCR.CCK8 was used to detect cell proliferation of HL-60 cells.Flow cytometry was used to detect apoptosis of HL-60 cells.ADM-treated HL-60 cells and CCK8 were used to detect the sensitivity of HL-60 cells to ADM.The target relationship between miR-424 and MEK1 was detected by double luciferase reporter gene.Western blot was used to detect the relative expression levels of MEK1,Ki67,Bcl-2 and Bax proteins.Results Compared with the normal control group,the relative expression level of miR-424 in HL-60 cells in the blank control group and NC group decreased significantly,and the relative expression level of MEK1 mRNA increased significantly(P<0.05).Compared with the blank control group and NC group,the proliferation rate of HL-60 cells,ADM IC50,MEK1 mRNA,the relative expression levels of Ki67,Bcl-2 and MEK1 proteins in miR-424 mimic group were significantly reduced,and the expression of miR-424,apoptosis rate,and relative expression level of Bax protein were significantly increased(P<0.05).And the relative activity of luciferase in miR-424 mimic+WT-MEK13'-UTR group was lower than that in miR-424 NC+WT-MEK13'-UTR group(P<0.05).Conclusion The expression of miR-424 is low in leukemia HL-60 cells.Overexpression of miR-424 may target to inhibit MEK1 expression,inhibit proliferation and promote apoptosis of HL-60 cells,and improve the sensitivity of cells to adriamycin.