标题：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

      作者：戴进前，倪庆仁，张韵洁，任婧婧，李光，张晓波，宋艳萍    西安市中心医院西安市血液病研究所，陕西 西安 710003

      卷次： 2021年32卷14期

      【摘要】 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-MEK1 3'-UTR组的荧光素酶相对活性低于miR-424 NC+WT-MEK1 3'-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。
      【关键词】 微小RNA-424；白血病HL-60细胞；丝裂原活化蛋白激酶1；增殖；凋亡；阿霉素耐药性
      【中图分类号】 R733.7 【文献标识码】 A 【文章编号】 1003—6350（2021）14—1773—05

Effects of MEK1 targeting miR-424 on proliferation, apoptosis, and adriamycin resistance of leukemia HL-60cell line.
DAI Jin-qian, NI Qing-ren, ZHANG Yun-jie, REN Jing-jing, LI Guang, ZHANG Xiao-bo, SONG Yan-ping.Department of Hematology, Xi'an Central Hospital, Xi'an Institute of Hematology, Xi'an 710003, Shaanxi, CHINA
【Abstract】 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 bloodmonocytes (PBMC) and human myeloid leukemia cells HL-60 were cultured in vitro, and HL-60 cells were transfectedwith miR-424 negative control and miR-424 mimic. The cells were divided into blank control group, NC group, andmiR-424 mimic group, and PBMC were used as normal control group. The relative expression levels of miR-424, mito-gen-activated protein kinase kinase 1 (MEK1) mRNA in PBMC and HL-60 cells were detected by qRT-PCR. CCK8 wasused to detect cell proliferation of HL-60 cells. Flow cytometry was used to detect apoptosis of HL-60 cells. ADM-treat-ed HL-60 cells and CCK8 were used to detect the sensitivity of HL-60 cells to ADM. The target relationship betweenmiR-424 and MEK1 was detected by double luciferase reporter gene. Western blot was used to detect the relative expres-sion levels of MEK1, Ki67, Bcl-2 and Bax proteins. Results Compared with the normal control group, the relative ex-pression level of miR-424 in HL-60 cells in the blank control group and NC group decreased significantly, and the rela-tive expression level of MEK1 mRNA increased significantly (P<0.05). Compared with the blank control group and NCgroup, the proliferation rate of HL-60 cells, ADM IC50, MEK1 mRNA, the relative expression levels of Ki67, Bcl-2and 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 luciferasein miR-424 mimic+WT-MEK1 3'-UTR group was lower than that in miR-424 NC+WT-MEK1 3'-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 in-hibit MEK1 expression, inhibit proliferation and promote apoptosis of HL-60 cells, and improve the sensitivity of cellsto adriamycin.
      【Key words】 MicroRNA-424; Leukemia HL-60 cells; Mitogen-activated protein kinase 1; Proliferation; Apopto-sis; Adriamycin resistancedoi:10.3969/j.issn.1003-6350.2021.14.002