CAJ | 학술논문

目的研究微小RNA 125b (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HL-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(Bak1)很可能是白血病细胞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Bak1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Bak1报告基因载体没有抑制作用.Western blot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Bak1的表达(P＜0.01).结论 Bak1是miR-125b在人AML中的一个靶基因.miR-125b很可能通过抑制Bak1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.
목적 연구미소RNA 125b (miR-125b)재인급성수계백혈병(AML)중적작용,병탐토기대파기인적조공궤제.방법 채용CCK-8계수법검측miR-125b대인AML세포계NB4、HL-60급인배신세포계HEK-293T증식적영향.용생물신식학방법예측miR-125b촉진세포증식적잠재상관파기인,구건용우감정miR-125b파기인적보고기인재체,병여miR-125b소분자유사물공전염293T세포,검측보고기인재체중형광소매활력.재NB4화HL-60세포중험증miR-125b여기파기인적관계.결과 miR-125b능현저촉진인NB4화HL-60세포악성증식,여대조조상비차이유통계학의의(P＜0.05);이대293T세포몰유명현영향.재선연건예측발현촉조망기인Bcl-2길항인자1(Bak1)흔가능시백혈병세포miR-125b잠재적1개파기인;쌍형광보고실험결과표명miR-125b능현저지억제함3'-UTR적Bak1보고기인활성,형광소매활성하강53.8％(P＜ 0.01),이대함 3'--UTR돌변위점적Bak1보고기인재체몰유억제작용.Western blot검측결과현시NB4화HL-60세포과표체miR-125b,병현저억제내원성Bak1적표체(P＜0.01).결론 Bak1시miR-125b재인AML중적일개파기인.miR-125b흔가능통과억제Bak1표체래촉진인AML세포악성증식,종이재AML발병중기유사“암기인”적작용.
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 plasmid 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-antagonist/killer 1 (Bak1),a pro-apoptotic gene,was a target gene of miR-125b by software predicts.Reporter vector containing the 3'-UTR Bak1 wild and mutation sites were co-transfected with small molecule analogues of miR-125b in HEK-293T cells.Dual luciferase reporter gene assay system showed that miR-125b significantly suppressed the reporter gene activity containing Bak1 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 Bak1 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 Bak1,so we speculated that miR-125b as an oncogene could be a potential therapeutic target for treating AML.