miR-142-3p靶向调控mll-af4融合基因的实验研究  

Targeted Regulation of mll-af4 Fusion Gene by miR-142-3p

作　　者：窦立萍[1] 李永辉[1] 徐诚望[1] 王莉莉[1] 于力[1] 

机构地区：[1]解放军总医院血液科,北京100853

出　　处：《中国实验血液学杂志》2010年第6期1595-1599,共5页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号30800482;90919044);973;国家重点基础研究发展计划(编号2005CB522400);首都医学发展科研基金(编号2007-2040)

摘　　要：本研究探讨mll-af4融合基因的表观遗传学调控机制,以筛选靶向调控mll-af4融合基因的microRNA。利用Targetscan在线分析软件预测特异性靶向mllaf4基因3′端非翻译区域(3′-untranslated region,3′UTR)的microRNA,采用PCR方法从1例健康供者DNA中扩增mll-af4基因3′UTR序列,插入经EcoRI和PstI双酶切的荧光素酶报告载体pGL3-M,采用脂质体SuperFect包裹荧光素酶重组质粒及microRNA表达质粒转染293T细胞,应用双荧光素酶检测试剂盒测定荧光素酶活性。miR-142-3p mimics以脂质体Hiperfect转染至RS4;11细胞,选用实时定量PCR及Western blot检测mll-af4 mRNA及蛋白的表达。结果表明,成功构建了含有1935bp、2104bp和1371bp的mll-af4基因3′UTR序列的荧光素酶报告重组质粒pGL3-AF4-3′UTR,并通过酶切及基因测序方法鉴定得到证实。荧光素酶报告实验提示,miR-142组荧光素酶活性明显低于对照组。RS4;11细胞中过表达miR-142-3p可以明显下调MLL-AF4融合蛋白及mRNA表达。结论:miR-142-3p通过靶向结合mll-af4基因3′UTR的结合位点特异调控mll-af4基因表达。In order to analyze the possible epigenetic regulation mechanism of mll-af4 gene expression and find the possible microRNA regulating mll-af4 gene expression, targetscan software was used to analyze potential microRNA target sites in 3′-UTR of mll-af4. 3′-UTR fragment of mll-af4 was amplified by PCR. PCR products were cloned into EcoR I /Pst I-digested pGL3-M reporter vector, placing the 3′-UTR with potential microRNA binding site downstream of coding sequence of luciferase. The construct was cotransfected in 293T cells with control plasmid or plasmids expressing microRNAs regulating mll-af4 potentially. Western blot and RT-PCR were used to detect the expression level of mll-af4 protein and mRNA in RS4;11 cells after transfection of miR-142-3p, respectively. The results showed that the pGL3-AF4-3′UTR of luciferase reporter recombinant plasmid containg the 3′UTR sequence of mll-af4 gene with 1935 bp, 2104 bp and 1371 bp was constructed successfully and was confirmed by enzyme digestion and gene sequencing. The luciferase assay revealed that overexpression of miR-142 could reduce the luciferase activity from the reporter construct containing the mll-af4 3′-UTR significantly. Protein and mRNA expressions of mll-af4 were found to be downregulated by miR-142-3p. It is concluded that miR-142-3p regulates the expression of mll-af4 through target binding the mll-af4 gene 3′UTR site.

关 键 词：MICRORNA 白血病 mll-af4融合基因 miR-142-3p 

分 类 号：R733.71[医药卫生—肿瘤]