Figure 5

The pri- miR- 221/ 222 transcript overexpressed in AML is at least 23 kb long. A) Deep sequencing data and chromatin marks in the relevant region in chromosome band Xp11.3 indicate that 5.6 kb, 28.2 kb, and/or 108.5 kb pri-miR-221/222 transcripts may exist. UCSC genome browser screenshot of chrX: 45,420,000-45,650,000, hg18, showing RNA-seq data from Hs27 foreskin fibroblasts (Vlatkovic IM, unpublished results), GRO-Seq data from IMR90 lung fibroblasts (red, forward, blue, reverse strand) [42], ENCODE histone modification ChIP-seq data for H3K4me3 (orange) and H3K36me3 (brown) in GM12878, K562, H1-hESC, HepG2, HUVEC, and HMEC cells [51], the positions of mature miR-221 and miR-222, the positions of primers used for qRT-PCR, and the putative pri-miR-221/222 transcripts (arrows). B-F) qRT-PCR on 3 blast-enriched AML and 2 healthy control PB samples using primers surrounding mature miR-221 (B), primers near the predicted 5’-ends of the 5.6 kb (C), 28.2 kb (D), and 108.5 kb (E) transcripts, and near the 3’ end common to all predicted transcripts (F). Transcript levels were determined by qRT-PCR and normalized to those of the housekeeping gene ß-2-microglobulin using the ΔΔct method [40] and PB from donor A (marked with #) as a calibrator. Note the differences in scale of the y-axis between the different panels. G) Luciferase assays demonstrate transcription activating potential of a region near the predicted start site of the 28.2 kb transcript. pGL3-P(-1874/+45), pGL3-P(+17/+1952), and the parental vector pGL3-P were transiently transfected into MCF7 cells, and luciferase activities were determined 2 days later. To control for transfection efficiency, firefly luciferase activity was normalized to renilla luciferase activity expressed from a cotransfected plasmid. Data represent mean +/- SEM from three biological replicates.