Analysis of the miR-183/-96/-182 cluster promoter region. (A) ENCODE project analysis of the upstream sequence of the miR-183/-96/-182 cluster: sequences in the red box represent the region from 5054 bp to 9324 bp upstream of the human miR-183 precursor that is highly conserved and enriched for 3K27Ac histone marks. (B) Fragmental reverse transcription (RT)-PCR demonstrated that the miR-183/-96/-182 cluster was transcribed in the same pri-miRNA: upper panel shows a schematic representation of the location of RT-PCR fragments and the miR-183/-96/-182 cluster in chromosome; lower panel shows the RT-PCR results of MCF-10A, MCF-7 and T47D cell cDNAs. Genomic DNA of MCF-7 cell was used as a positive control to check the efficiency of primer pairs; RNA sample, which did not undergo the reverse transcription reaction, was used as a negative control. (C) Luciferase assay indicated that most active regulatory elements were located within 1 kb from upstream of the TSS region of miR-183/-96/-182 cluster. All luciferase activities were normalized to those obtained with the pGL3-Basic vector alone. Error bars represent SD (n = 4).