a novel vector-based method for exclusive overexpression of star-form micrornas一种新颖的基于矢量的独家star-form超表达的小分子核糖核酸的方法.pdfVIP

A Novel Vector-Based Method for Exclusive Overexpression of Star-Form MicroRNAs Bo Qu1,2., Xiao Han1,2., Yuanjia Tang1,2, Nan Shen1,2,3* 1Joint Molecular Rheumatology Laboratory of the Institute of Health Sciences and Shanghai Renji Hospital, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China, 2 Key Laboratory of Stem Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China, 3 Division of Rheumatology and the Center for Autoimmune Genomics and Etiology (CAGE), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America Abstract The roles of microRNAs (miRNAs) as important regulators of gene expression have been studied intensively. Although most of these investigations have involved the highly expressed form of the two mature miRNA species, increasing evidence points to essential roles for star-form microRNAs (miRNA*), which are usually expressed at much lower levels. Owing to the nature of miRNA biogenesis, it is challenging to use plasmids containing miRNA coding sequences for gain-of-function experiments concerning the roles of microRNA* species. Synthetic microRNA mimics could introduce specific miRNA* species into cells, but this transient overexpression system has many shortcomings. Here, we report that specific miRNA* species can be overexpressed by introducing artificially designed stem-loop sequences into short hairpin RNA (shRNA) overexpression vectors. By our prototypic plasmid, designed to overexpress hsa-miR-146b-3p, we successfully expressed high levels of hsa-miR-146b-3p without detectable change of hsa-miR-146b-5p. Functional analysis involving luciferase reporter assays showed that, like na