MicroRNA-3666调节白血病细胞PTEN基因的表达

MicroRNA-3666 regulates expression of phosphatase and tensin homologue deleted on chromosome ten in leukemic cells

目的:探讨白血病细胞中microRNA-3666(miR-3666)对第10号染色体缺失的磷酸酶和张力蛋白同源基因(PTEN)表达的调控作用。方法:qRT-PCR检测miR-3666在正常人外周血单个核细胞、不同类型白血病细胞系以及临床初诊未治的不同类型白血病细胞中的表达差异,利用TargetScan在线分析软件预测miR-3666潜在靶基因PTEN后,构建含有靶基因野生型3’端非翻译区域(3’UTR)及突变型3’UTR(缺失了整段预测的miR-3666结合序列)的双萤光素酶报告基因质粒,采用脂质体Lipofectamine 2000包裹双萤光素酶重组质粒及miR-3666模拟体或阴性对照,共转染HEK293T细胞,应用双萤光素酶检测试剂盒测定萤光素酶活性。FAM标记的miR-3666抑制体和阴性对照分别核转染至K562细胞,FACS检测转染效率,并采用Western blotting检测PTEN蛋白的表达。结果:miR-3666在人白血病细胞系及原代白血病细胞中的表达明显高于正常人外周血单个核细胞,尤其高表达于K562细胞系及原代慢性粒细胞白血病细胞中;双萤光素酶报告基因测定系统验证PTEN是miR-3666的潜在靶基因;K562细胞中下调miR-3666后,Western blotting检测结果显示PTEN蛋白显著上调。结论:白血病细胞中miR-3666的表达上调,下调miR-3666后可以促进靶基因PTEN的表达,miR-3666有可能成为白血病治疗的新靶点。

AIM ： To explore the regulatory effect of microRNA-3666 （miR-3666） on the expression of its tar- get gene phosphatase and tensin homologue deleted on chromosome ten （PTEN） in leukemic cells. METHODS： miR- 3666 expression levels in normal peripheral blood mononuclear cells and leukemic cells were determined by quantitative real-time PCR. miR-3666 targeting PTEN 3＇-untranslated region （3＇UTR） was predicted by TargetScan software. 3＇UTR of PTEN was inserted in the dual luciferase reporter vector psiCHECK2. The reporter activity was evaluated by the Dual-Lu- ciferase Reporter Assay System after the luciferase promoter vector and miRNA were co-transfected into HEK293T cell line. K562 cells were transfected with synthetic miR-3666 inhibitor （anti-miR-3666） or a synthetic control miRNA （anti-miR- C）. The expression of PTEN protein in the above transfected K562 ceils was determined by Western blotting. RESULTS： miR-3666 was up-regidated in the human leukemic cell lines and primary leukemic cells compared to normal peripheral blood mononuclear cells. The results of dual luciferase assays validated PTEN as a specific target gene of miR-3666. Inhi- bition of miR-3666 resulted in an up-regulation of PTEN protein expression in the K562 ceils. CONCLUSION： miR-3666 is over-expressed in leukemic cells. The abnormal over-expression of miR-3666 may play a key role in leukemia due to the down-regulation of PTEN.