miR-125b靶向抑制Bak1表达对人髓系白血病细胞增殖的影响

miR-125b promotes proliferation of human acute myeloid leukemia cells by targeting Bakl

目的研究微小RNAl25b（miR-125b）在人急性髓系白血病（AML）中的作用，并探讨其对靶基因的调控机制。方法采用CCK-8计数法检测miR-125b对人AML细胞系NB4、HL-60及人胚肾细胞系HEK-293T增殖的影响。用生物信息学方法预测miR-125b促进细胞增殖的潜在相关靶基因，构建用于鉴定miR-125b靶基因的报告基因载体，并与miR-125b小分子类似物共转染293T细胞，检测报告基因载体中荧光素酶活力。在NB4和HL-60细胞中验证miR-125b与其靶基因的关系。结果miR-125b能显著促进人NB4和HE-60细胞恶性增殖，与对照组相比差异有统计学意义（P〈0．05）；而对293T细胞没有明显影响。在线软件预测发现促凋亡基因Bcl-2拮抗因子1（Bakl）很可能是门血病细胞miR-125b潜在的1个靶基因；双荧光报告实验结果表明miR-125b能显著地抑制含3＇-UTR的Bakl报告基因活性，荧光素酶活性下降53．8％（P〈0．01），而对含3＇-UTR突变位点的Bakl报告基因载体没有抑制作用。Westernblot检测结果显示NB4和HL-60细胞过表达miR-125b，并显著抑制内源性Bakl的表达（P〈0．01）。结论Bakl是miR-125b在人AML中的一个靶基冈。miR-125b很可能通过抑制BakI表达来促进人AML细胞恶性增殖，从而在AML发病中起类似“癌基因”的作用。

Objective To investigate miR-125b regulation mechanism by identifying miR-125b target genes and its function in acute myeloid leukemia（AML）. Methods The bioinformatics software and database were applied to predict and analyze target genes of miR-125b. The vector contained the target gene 3＇-UTR portion cloned into a luciferase reporter construct. A luciferase reporter assay was performed following co-transfection of small molecular miR- 125b mimics and target gene wild-type or mutant plas- mid into HEK-293T cells. Further in leukemia cell lines NB4 and HL-60, the protein level of target gene was measured by Western blot after overexpression miR-125b. Finally, the viabilities of NB4 and HL-60 cells were measured by CCK-8 assay at 24 h, 48 h, 72 h, 96 h after electroporation. Results Bcl-2-antago- nist/killer 1 （Bakl）, a pro-apoptotic gene, was a target gene of miR- 125b by software predicts. Reporter vector containing the 3＇-UTR Bakl wild and mutation sites were co-transfected with small molecule ana- logues of miR-125b in HEK-293T cells. Dual luciferase reporter gene assay system showed that miR-125b significantly suppressed the reporter gene activity containing Bakl 3＇-UTR by about 53.8% （P〈0.05）, but it didn＇t suppresse the reporter gene activity containing 3＇-UTR Bakl mutation. Western blot showed that miR-125b mimics significantly down-regulated the expression of Bakl in human leukemia cell lines NB4 and HL-60. Meanwhile, the growth rate of cells treated with miR-125b obviously increased compared with that in control by CCK-8 test （P 〈 0.05）. Conclusion Our findings strongly indicated that BAK1 was a downstream target gene of miR-125b, and miR-125b promoted proliferation in human AML cells at least partially by targeting Bak 1, so we speculated that miR-125b as an oncogene could be a potential therapeutic target for treating AML.