目的： 研究三氧化二砷（As 2O 3）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、As 2O 3组、pmiR-203组、空质粒对照组、As 2O 3联合空质粒对照组和As 2O 3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As 2O 3使K562细胞对As 2O 3的敏感性提高到单用As 2O 3的4.86倍，IC50 从3.4 μmol/L降低至0.7 μmol/L，两者联用表现为协同作用。As 2O 3联合pmiR-203组的细胞凋亡率显著高于As 2O 3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As 2O 3的敏感性，miR-203和As 2O 3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 (As 2O 3) 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 As 2O 3 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 As 2O 3 treatment increased the sensitivity K562 cells to As 2O 3 by up to 4.86-fold alone while the IC 50 was decreased from 3.4 μmol/L to 0.7 μmol/L as compared with As 2O 3. The number of apoptotic cells was increased in pmiR-203-transfected K562 cells treated with As 2O 3 (\[29.97±3.19\]%) compared with As 2O 3 alone (\[10.77±1.71\]%,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 As 2O 3, at least partially through down-regulation of Bcr/abl expression.

广西自然科学基金资助项目（2012GXNSFAA053177）