Fig. 4

Loss of miR 2355-5p reduces cell survival and proliferation. (A) Schematic representation of the genomic location targeted by three CRISPR/Cas9 gRNAs to repress miR-2355-5p. (B) RT-qPCR quantification of miR-2355-5p in 786-0, 786-0 Cr.Control (CTRL), 786-0 Cr.2355–2 A, 786-0 Cr.2355-2B, and 786-0 Cr.2355–2 C cells (N = 3). (C) RT-qPCR quantification of miR-2355-5p in A498, A498 Cr.CTRL, A498 Cr.2355–2 A, and A498 Cr.2355-2B cells (N = 3). E-G Image illustration (D-E) and relative number of colonies (F-G) measured by clonogenic assays in 786-0 Cr.2355 and A498 Cr.2355 clones compared to appropriate controls (N = 4). H-I Cell proliferation measured by daily cell counts in 786-0 Cr.2355 (H) and A498 Cr.2355 (I) clones compared to appropriate control cells (N = 4). J-K Flow cytometry analysis and quantification of 786 Cr.2355 models and control cells following PI staining. MiRNA expression was normalized with RNU44. The data are presented as the Mean ± SEM. Statistical analysis was performed using two-tailed Student’s t-tests (B, C,F, G) or two-way ANOVA with Dunnett’s multiple comparison test compared to 786 Cr.CTRL (H, J) or A498 Cr.CTRL (I). (ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)