Several miRNAs are highly upregulated in premalignant tPTEN^−/− DP thymocytes. (A) Heat-map representing miRNAs with a combined P value of < .05 and a δ log median ratio >0.2. Forty-three of 599 miRNAs evaluated were differentially expressed in tPTEN^−/− DP thymocytes from 3 premalignant 9-week-old mice compared with 3 littermate controls (129/SvJ × CBA × C57BL/6 background). Red and blue indicate miRNA expression levels above and below the mean, respectively. (B) Quantitative RT-PCR validation of the miRNA array results on fluorescence-activated cell sorting (FACS)-sorted DP and CD4 SP thymocytes and column-purified mature T cells from 5-week-old and premalignant 9-week-old mice. miRNA expression levels are normalized to miR-16, which is expressed similarly in WT and tPTEN^−/− T-cell subsets (supplemental Figure 1B). Fold change is relative to WT DP thymocytes. (C) Quantitative RT-PCR analysis of the correlation between miRNA and c-myc expression levels in 9-week-old tPTEN^−/− premalignant DP thymocytes. Fold change is relative to littermate WT DP thymocytes. c-myc expression is represented as less than or greater than a twofold increase over WT levels. (D) Quantitative RT-PCR analysis of c-myc expression in DP and CD4 SP thymocytes and mature T cells from premalignant 9-week-old mice with a pattern of miR-146a and miR-146b expression as shown in (B). (E) miR-146a and miR-146b expression levels in thymocytes and lymph node tumor cells from 12-week-old mice (129/SvJ × CBA × C57BL/6 background). Fold change is relative to WT DP thymocytes. Data in (B) and (D-E) are presented as mean ± SD and are representative of 3 or more independent experiments. Statistics were calculated by Student t test (***P < .001, **P < .01, *P < .05, n.s. not significant).