microRNA-1271靶向调控白血病细胞CYLD蛋白的表达  

microRNA- 1271 regulates the expression of CYLD in leukemia cells

作　　者：张茜[1] 王媛媛[1] 刘丽萍[1] 倪芳[2] 

机构地区：[1]安徽医科大学第二附属医院药剂科,合肥230601 [2]安徽医科大学基础医学院,合肥230032

出　　处：《安徽医科大学学报》2015年第7期908-911,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年项目(编号:31300715);安徽省自然科学基金青年项目(编号:1308085QH136)

摘　　要：目的探讨人白血病细胞中微小RNA(microRNA)-1271对CYLD蛋白表达的调控作用。方法 qRT-PCR检测microRNA-1271在不同的人白血病细胞系、临床初诊未治的几种类型原代白血病细胞、正常人外周血单个核细胞中的表达差异,利用Targetscan信息学预测软件预测microRNA-1271靶向CYLD基因,构建携带靶基因野生型及突变型3’非翻译区(3’UTR)(缺失了整段预测的microRNA-1271结合序列)的双荧光素酶报告基因质粒,采用脂质体Lipofectamine 3000包裹双荧光素酶重组质粒及microRNA-1271模拟物(mimic)或阴性对照,共转染HEK293A细胞,应用双荧光素酶报告基因检测试剂盒测定荧光素酶活性。FAM标记的microRNA-1271抑制物和阴性对照分别核转染至人白血病细胞系K562细胞,Western blot法检测CYLD蛋白水平的表达变化。结果 microRNA-1271在不同人白血病细胞系以及临床初诊未治的原代白血病细胞中的表达均明显高于正常人外周血单个核细胞,双荧光素酶报告基因实验验证CYLD是microRNA-1271的潜在靶基因;K562细胞中转染microRNA-1271抑制物下调microRNA-1271后,Western blot法检测结果显示CYLD蛋白水平明显上调。结论人白血病细胞中microRNA-1271的表达上调,下调microRNA-1271后可以促进靶基因CYLD蛋白的表达,microRNA-1271有可能成为白血病治疗的新靶点。Objective To explore the regulatory effect of microRNA-1271 on the expression of its target gene CYLD in leukemia cells. Methods microRNA-1271 expression levels in normal peripheral blood mononuclear cells（ PB-MC） and leukemia cells were determined by quantitative real-time PCR. microRNA-1271 targeting CYLD 3′-UTR was predicted by TargetScan. 3′-UTR of CYLD was inserted into the dual luciferase reporter vector psi-CHECK2. The reporter activity was evaluated by the dual Luciferase Reporter Assay System after the luciferase promoter vector and microRNA were co-transferred into the HEK293A cell line. K562 cell lines were transfected with microRNA-1271 inhibitors （ anti-microRNA-1271 ） or a negative control microRNA （ anti-microRNA-NC ） , the expression of CYLD protein in the above transfected K562 cells were determined by Western blot. Results microRNA-1271 was up-regulated in human leukemia cell lines and primary leukemia cells compared to normal human PBMCs. The re-sults of dual luciferase assays validate CYLD as a specific target gene of microRNA-1271 . Inhibition of microRNA-1271 resulted in the upregulation of CYLD protein expression in K562 cell line. Conclusion microRNA-1271 is overexpressed in leukemia cells, the microRNA-1271 abnormal overexpression may play a key role in leukemia due to the down-regulation of CYLD.

关 键 词：白血病 MICRORNA microRNA-1271 CYLD 

分 类 号：R363[医药卫生—病理学]