miR-196a靶向调控HOXA9基因的实验研究

HOXA9 is direct target of miR-196a

目的:研究HOXA9基因的表观遗传学调控机制,筛选靶向调控HOXA9基因的microRNA。方法:利用Tar-getscan在线分析软件预测特异性靶向HOXA9基因3'端非编码区域(3’UTR)的microRNA,采用PCR方法从1例健康供者DNA中扩增HOXA9基因3’UTR序列,插入经XbaI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。构建miR-196a结合位点突变的HOXA9基因3’端非编码区域突变型荧光素酶报告载体,采用脂质体SuperFect包裹荧光素酶突变型重组质粒及miR-196a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-196amimics以脂质体Hiperfect转染至MV4-11细胞,实时定量PCR及Westernblot检测HOXA9mRNA及蛋白的表达。结果:成功构建了含有1628bp的HOXA9基因3’UTR序列的荧光素酶报告重组质粒pGL3-HOXA9-3’UTR,并通过酶切及基因测序方法的鉴定;荧光素酶报告实验提示miR-196a组荧光素酶活性明显低于对照组。成功构建了miR-196a的2个结合位点突变的HOXA9基因3’UTR序列的突变型荧光素酶报告重组质粒pGL3-HOXA9-mut3’UTR,并通过基因测序方法的鉴定。miR-196a可以下调野生型质粒的荧光素酶活性,不影响突变型质粒的荧光素酶活性。MV4-11细胞中过表达miR-196a,可以明显下调HOXA9mRNA及蛋白表达。结论:miR-196a通过靶向结合HOXA9基因3’UTR的结合位点特异调控HOXA9基因。

AIM： To analyze the possible epigenetic regulation mechanism of HOXA9 gene expression and find the possible microRNA regulating HOXA9 gene expression. METHODS： Targetscan software was used to analyze potential microRNA target sites in 3 ＇-UTR of human HOXAg. 3＇-UTR fragment of HOXA9 was amplified by PCR. PCR products were cloned into Xba I/Pst I-digested pGL3-M reporter vector, placing the 3＇-UTR with potential microRNA binding sites downstream of coding sequence of luciferase. Mutant 3＇UTRs were generated by overlap extension PCR method. The construct was cotransfected in 293T cells with control plasmid or plasmids expressing microRNAs regula- ting HOXA9 potentially. Western blot and RT-PCR were used to detect the expression level of HOXA9 protein and mRNA in MV4-11 cells after transfection of miR196a. RESULTS： The results of luciferase assays revealed that overexpression of miR-196a could reduce the luciferase activity from the reporter construct containing the HOXA9 3 ＇-UTR significantly. The activity of the reporter construct mutated at the specific miR-196a target site was unaffected. Protein and mRNA of HOXA9 was found to be downregulated by miR-196a. CONCLUSION： Theses results suggested that miR-196a regulates the expression of HOXA9 by targeting the complementary sites.