三氧化二砷联合miR-203对人白血病K562细胞的抑制作用及其机制  

Inhibiting effects of arsenic trioxide in combination with miR-203 on leukemic K562 cells and its mechanisms

作　　者：黄之虎[1] 韦思羽[1] 农朝赞[1] 韦仕喻[1] 农少云[1] 郭凌霄[1] 李育敏[1] 何金花 杨林杰[3] 

机构地区：[1]广西壮族自治区民族医院检验科,广西南宁530001 [2]广州市番禺区中心医院检验科,广东广州511400 [3]广州医科大学附属第二医院神经科学研究所,广东广州510260

出　　处：《中国肿瘤生物治疗杂志》2014年第5期499-504,共6页Chinese Journal of Cancer Biotherapy

基　　金：广西自然科学基金资助项目(2012GXNSFAA053177)~~

摘　　要：目的:研究三氧化二砷(As2O3)联合过表达的微小RNA-203(microRNA-203,miR-203)对白血病K562细胞的抑制作用及其可能的分子机制。方法:将miR-203的真核表达载体pmiR-203转染K562细胞,Real-time PCR检测细胞内miR-203的表达。将K562细胞分为空白对照组、As2O3组、pmiR-203组、空质粒对照组、As2O3联合空质粒对照组和As2O3联合pmiR-203组,MTT法检测各组细胞增殖抑制率,流式细胞术仪检测细胞凋亡率,Western Blotting检测细胞内Bcr/abl蛋白的表达水平。构建Bcr/abl 3'UTR和Bcr/abl mut-3'UTR的双荧光素酶报告基因载体,将其与pmiR-203共转染K562细胞,通过荧光素酶活性分析判断miR-203是否与Bcr/abl基因的3'UTR结合。结果:miR-203的真核表达载体pmiR-203转染K562细胞后,细胞内miR-203的表达水平明显增高(P<0.05)。高表达miR-203联合As2O3使K562细胞对As2O3的敏感性提高到单用As2O3的4.86倍,IC50从3.4μmol/L降低至0.7μmol/L,两者联用表现为协同作用。As2O3联合pmiR-203组的细胞凋亡率显著高于As2O3联合空质粒对照组[(29.97±3.19)%vs(10.77±1.71)%,P<0.05]。过表达miR-203显著下调K562细胞内Bcr/abl蛋白的表达水平。Bcr/abl 3'UTR中带有明确的miR-203结合位点。结论:miR-203可提高白血病K562细胞对As2O3的敏感性,miR-203和As2O3联用对K562细胞具有协同抑制作用,其作用机制可能与miR-203直接下调Bcr/abl融合基因的表达有关。Objective： To study the anti-tumor effect of arsenic trioxide（ As2O3） in combination with overexpression of miR-203 on leukemic K562 cells. Methods： Eukaryotic expression vector of miR-203（ pmiR-203） was transfected into K562 cells. Transcript levels of miR-203 were quantified by real-time quantitative PCR. K562 cells were incubated with different concentrations of As2O3 alone or in combination with pmiR-203. Cell viability was measured by MTT assay. Cell apoptosis was analyzed using flow cytometry. Bcr /abl protein contents were assessed by Western blotting. The ability of miR-203 to bind to Bcr /abl 3＇ UTR was determined by Bcr /abl 3＇ UTR and Bcr /abl mut-3＇ UTR dual luciferase report vector assays. Results： Levels of miR-203 transcript were significantly increased in pmiR-203-transfected K562 cells.Overexpression of miR-203 combined with As2O3 treatment increased the sensitivity K562 cells to As2O3 by up to 4. 86-foldalone while the IC50 was decreased from 3. 4 μmol /L to 0. 7 μmol /L as compared with As2O3. The number of apoptotic cells was increased in pmiR-203-transfected K562 cells treated with As2O3（ [29. 97 ±3. 19]%） compared with As2O3alone（ [10. 77 ± 1. 71]%,P〈 0. 05）. Overexpression of miR-203 resulted in decreases in Bcr /abl protein levels and Bcr /abl-3＇UTR reporter activity without affecting activity of Bcr /abl-mut-3＇UTR reporter in K562 cells. A binding site in the sequence of Bcr /abl-mut-3＇UTR was identified. Conclusion： Overexpression of miR-203 may enhance the sensitivity of leukemic K562 cells to As2O3,at least partially through down-regulation of Bcr/abl expression.

关 键 词：三氧化二砷 微小RNA-203 白血病 K562细胞 BCR/ABL 

分 类 号：R733.7[医药卫生—肿瘤] R730.5[医药卫生—临床医学]