Optimization of a microRNA expression vector for function analysis of microRNA.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs endogenously expressed in a tissue-type specific pattern. Recent studies have demonstrated that miRNAs are involved in almost all cellular biological processes, including cellular development, differentiation, apoptosis, and proliferation. To elucidate the function of miRNAs in biological processes, it is crucial that we develop miRNA-expressing vectors for the efficient expression of miRNAs in cultured cells and animals. At the present time, however, no fully optimized miRNA-expressing vectors have been developed, since such vectors require consideration of the choice of promoters and several other complex factors. In this study, we constructed various types of plasmid vectors expressing human miR-199a. There are two genes encoding miR-199a in the different chromosomes, resulting in expression of two different precursors which produce the two mature miRNAs from the 5'- and 3'-strands (miR-199a-5p and -3p). When the miR-199a precursors containing the genomic sequences flanking the hairpin were expressed, the cytomegalovirus (CMV) promoter, CMV promoter/enhancer containing the intron A (CMVi), and CMV enhancer/β-actin (CA) promoter were more effective than the human phosphoglycerate kinase (PGK) promoter. The reduction levels by the human U6 promoter-transcribed miR-199a were different between the cell lines. The suppressive effects of miR-199a on the reporter gene expression were different between miR-199a-5p and -3p, especially when miR-199a was expressed as a stem-loop structure under the control of the U6 promoter. Expression of miR-199a as a short-hairpin RNA (shRNA) with an artificial hairpin sequence and independent expression of the mature miR-199a and its complementary strand were effective for distinguishing the function of the 5'- and 3'-strand of the miRNA. In addition, expression of miRNAs as an shRNA and separate expression of mature miRNAs and their complementary strand would be promising methods under conditions in which the processing steps of miRNAs are impaired. The results of this study provide important information on the construction of miRNA-expressing vectors for miRNA function analysis as well as for gene therapy using miRNA-expressing vectors.