A Novel Anti-Apoptotic Role for Mir-290-295 Figure 4. Downregulation of Casp2 and Ei24 partially rescues the increased apoptotic rate of Dcr KO cells following genotoxic stress. A. The percentage of cells expressing cleaved Caspase 3 (Casp3) in Dcr WT and Dcr KO ES cells after exposure to 5-Gy radiation (0 and 24 hours after radiation treatment). Cleaved Casp3 was assayed by flow cytometry, and was used to estimate apoptotic response. Apoptosis rate of Dcr KO cells is shown in black bars, and that of WT cells is shown in white bars. B. Dcr KO cells were treated with 5-Gy radiation 24 hours after transfection of 50 nM miR-295, miR-290-3p, Bim siRNA, Casp2 siRNA or Ei24 siRNA. Casp3 activity was assayed 0 and 24 h after the treatment, and the difference in apoptosis rate is shown. Transfection of seed mutants and control siRNAs into Dcr KO cells, and transfection of control siRNAs into WT cells served as controls. C,D. A similar series of experiments was performed in Dcr WT and Dcr KO cells using 100 nM doxorubicin. n$3 for all experiments. Results are shown as mean 6 S.E.M. P-values were obtained by Mann-Whitney tests, * denotes p#0.05, and ** denotes p#0.01. doi:10.1371/journal.pgen.1002054.g004 KO cells exhibited Casp3 activity (Figure 4A). Similar results were seen using Annexin V staining, a complementary assay for detecting early apoptosis (Figure S3C). Importantly, mature miRNA levels from the mir-295 cluster were unchanged by these stressors (Figure S3D). Therefore, it appears that loss of mature miRNAs leads to an enhancement of apoptosis in the presence of DNA damage. To examine whether the miR-295 targets modulated apoptosis, we transfected a series of siRNAs into Dcr KO cells and evaluated cell death following irradiation. The difference in Casp3 activation between 0 and 24 h timepoints was calculated in order to account for differences in transfection-specific toxicity (Figure S6A and S6B). Relative to control siRNAs, transfection of miR-290-3p or miR-295 drastically decreased the apoptosis response of Dcr KO cells to gamma irradiation (Figure 4B, Figure S6A and S6B). The reduction in apoptosis is specific to the AAGUGC seed, as seed mutants failed to rescue Dcr KO ES cells from apoptosis. When we applied siRNAs specific to each validated target, or to Bim, a well-characterized proapoptotic factor, cells exhibited a decrease of 5–10% in Casp3 activation 24 h after irradiation, a level similar to mir-295 cluster miRNA overexpression (Figure 4B, Figure S6A and S6B). Similar findings were obtained when cells were treated with 100 nM –300 nM doxorubicin, suggesting that the identified pathways are relevant to DNA damage in general (Figure 4C and 4D, Figure S4B, Figure S6C and S6D). Specific deletion of the mir-295 cluster enhances susceptibility to apoptosis upon DNA damage Because deletion of Dcr involves global miRNA loss, and three additional clusters containing the same or similar hexamer seed, mir-302, mir-467, and mir-17-92, are expressed in ESCs (Table S2), we examined the 295 KO line to determine the specific PLoS Genetics | www.plosgenetics.org 5 May 2011 | Volume 7 | Issue 5 | e1002054