Figure 3.

miR-19 miRNAs are essential for the oncogenic activity of mir-17-19b. (A) A schematic representation of the gene structural organization of mir-17-19b, mir-17-19b-Mut19, and miR-19b. 19a* and 19b* indicate miR-19 mutations. (B) miR-19 mutations specifically affected miR-19 expression in mir-17-19b. 3T3 cells were infected with MSCV-mir-17-19b, MSCV-mir-17-19b-Mut19, or control MSCV vectors. Expression levels of miR-17, miR-18a, miR-19a, miR-20a, and miR-19b were determined using TaqMan miRNA assays. miR-19 mutations specifically affected the expression of miR-19a and miR-19b, but not that of the adjacent mir-17-19b components. Error bars indicate SD (n = 3). (C) miR-19 is both necessary and sufficient for the oncogenic effect of mir-17-19b. Overexpression of miR-19b and mir-17-19b accelerated c-myc-induced lymphomagenesis to a similar degree, shown in Kaplan-Meier curves as the percentage of overall survival. Mutations in miR-19 greatly decreased the oncogenic activity of mir-17-19b in the Eμ-myc model. We compared the oncogenic effects of mir-17-19b, miR-19b, and mir-17-19b-Mut19 in the same adoptive transfer experiment. (D) miR-19 is both necessary and sufficient for the cell survival effect of mir-17-19b in vivo. Representative lymphomas from Eμ-myc, Eμ-myc/19b, and Eμ-myc/17-19b-Mut19 were stained for H&E and caspase-3, which indicated that the miR-19 mutations significantly compromised cell survival effects of mir-17-19b, while miR-19b overexrepssion suppresses apoptosis. The Eμ-myc/19b and Eμ-myc/17-19b-Mut19 tumors shown here were both IgM-negative B lymphomas. (Arrow) “Starry sky” feature of apoptotic tumor cells; (arrowhead) apoptotic cells with positive caspase-3 staining. Bar, 50 μm. (E) Quantification of caspase-3 staining of representative tumors from D as percentage of positive cells. For Eμ-myc/19b and Eμ-myc/17-19b-Mut19 tumors, only IgM-negative tumors were selected for this comparison (n = 3; error bars represent SEM).