Transgenic expression of mir-146a and mir-146b delays tumorigenesis in tPTEN^−/− mice. (A) miR-146a expression levels in total thymocytes from mir-146a transgenic line 8 (146a8) vs WT mice by RNase protection assay. miR-16, a miRNA that is abundant in thymocytes, was used as a reference for miRNA levels. (B) Quantitative RT-PCR analysis of miR-146a expression levels in FACS-sorted thymocyte and mature T-cell subsets from 146a8 transgenic and littermate WT control mice. Fold change is relative to WT DP thymocytes and normalized to miR-16. (C) Comparison of miR-146 expression levels in DP thymocytes from all characterized mir-146 transgenic founder lines. Fold change is relative to miR-146 expression in littermate WT controls. (D-E) Kaplan-Meier curves for the mir-146a transgenic lines 146a8 and 146a1 (D) and the mir-146b transgenic line (E) crossed to tPTEN^−/− mice on the C57BL/6 background. Statistical significance was calculated using the Gehan-Breslow-Wilcoxon method. (F) CD4 vs CD8 flow cytometric analysis of T-cell populations in the thymus and lymph nodes of 7-week-old 146b transgenic mice compared with littermate controls on both WT and tPTEN^−/− backgrounds. T-cell numbers are indicated on the lymph node plots to reflect the decrease in CD4 T-cell numbers with mir-146b transgene expression. Data are representative of 3 independent experiments. (G) Flow cytometric analysis of CD62L (l-selectin) surface expression in lymph node T cells from 7-week-old 146b transgenic and littermate control mice on WT and tPTEN^−/− backgrounds. Data are representative of 2 independent experiments.