miRNA expression. (A) Top 20 expressed miRNAs in AML1 or normal CD34 cells (total 25 miRNAs); the percentage of reads for each miRNA relative to the total number of miRNA reads is shown. (B) Top 10 miRNAs with the most increased expression in AML1 compared with normal CD34. (C) Top 10 miRNAs with the most reduced expression in AML1 compared with CD34.

isomiR pattern analysis. (A) miR-20a isomiRs. The reference sequence for miR-20a is shown with the mature miRNA bolded. Lower case letters represent nucleotide deviations from reference. The percentage of each miR-20a isomiR relative to the total sequence reads for miR-20a in the AML1 and CD34⁺ cells samples is shown. (B) The percentage of each isomiR relative to the total read counts for a specific miRNA were calculated for all miRNAs that had at least 1000 read counts between the AML1 and CD34 patient samples.

The TNFAIP2 3′-UTR mutation confers translation repression in a miRNA dependent fashion. (A) The sequence of the 3′-UTR of TNFAIP2 (14:102673002-102673041) flanking the A→G mutation at 14:102673033 is shown, along with predicted binding sites for miR-223 and miR-181b. (B) The region of the TNFAIP2 3′-UTR shown in panel A containing germline (TNFAIP2 germ) or mutated sequence (TNFAIP2 mut) was cloned downstream of Renilla luciferase (psi-check2 vector) and transiently transfected into K562, 293T, and HS-5 cells. Expression of Renilla luciferase relative to firefly luciferase (an internal control present in the psi-check2 vector) after normalization to the germline TNAIP2 3′-UTR signal is shown. (C) Expression of miR-223 and miR-181b relative to RNU48 is shown. (D) 293T or K562 cells were cotransfected with the TNFAIP2 3′-UTR Renilla luciferase reporter constructs and a vector directing high level expression of either miR-223 or miR-181b. Expression of Renilla luciferase relative to firefly luciferase is shown. (E) Representative Western blot showing DICER1 protein expression in parent KLE cells (Wt) or KLE cells expressing shRNA directed against the red fluorescent protein (shRFP) or DICER1 (shDcrA). The upper band is nonspecific. GAPDH was used as a loading control. (F) The shRFP and shDicer KLE cells lines were transiently transfected with the TNFAIP2 3′-UTR Renilla luciferase reporter constructs. Expression of Renilla luciferase relative to firefly luciferase is shown. Data represent the mean ± SEM of 3-5 independent experiments.

Identification of novel miRNAs. (A) Algorithm used to identify novel miRNAs. (B) Expression of novel miRNAs in the AML1 and normal CD34 samples. The percent contribution of each miRNA sequence to the total pool of known miRNA sequences was calculated by dividing individual miRNA read counts by the total number of miRNA sequence reads.

miR-223 expression in AML. (A) The relative expression of miR-223 in AML1 compared with the total expression of all miRNAs based on the Agilent array (array) and RNA sequencing (deep sequencing) data are shown. (B) The relative expression of miR-223 compared with RNU48 was determined using real time RT-PCR on undiluted RNA from AML1 and RNA diluted 10-fold (1/10) or 100-fold (1/100) with water. (C) miR-223 expression relative to RNU48 in 27 AML samples and 8 CD34 samples from healthy donors is shown. AML1 is indicated by the arrow. The dashed line indicates 2 SDs above the mean using data from the CD34 samples.

Deep sequencing of miRNA genes. All 695 miRNA genes present in the Sanger miRNA database at the time this study was initiated were PCR-amplified from leukemic genomic DNA from AML1 and subjected to 454-based sequencing. A total of 148 high confidence SNVs were identified, of which 108 were in dbSNPs. Analysis of previously generated whole genome sequence (WGS) data for the leukemic and skin (normal) genomes of AML1 showed that 13 novel germline SNPs were present in AML1. No somatic mutations were identified.