Figure 2

Let-7 binding to the NRAS 3′UTR mediates regulation of NRAS by circPVT1. (a) Schematic of the let-7 target protector masking let-7 binding sites in the NRAS 3′UTR. (b,c) Dual luciferase reporter assays using A549 cells co-transfected with (b) NRAS 3′UTR reporter vectors and let-7a-1 expression vector, or (c) NRAS 3′UTR reporter vector and either si-control or si-circPVT1. One-sample t-test of fold change against unity with Holm-Šidák correction for multiple comparisons. Additionally, in (c), ANOVA with Dunnett’s post-hoc test to compare si-circPVT1 set-ups to vector control. FL/RL, ratio of firefly and Renilla luciferase signals. (d,e) RT-qPCR measurements of endogenous (d) circPVT1 and (e) NRAS transcript levels in A549 cells upon co-transfection with either si-control or si-circPVT1 and either negative control target protector (TP-control) or let-7/NRAS 3′UTR target protector (TP-let-7). ANOVA with Dunnett’s post-hoc test. (f) Western blot for NRAS protein levels in A549 cells co-transfected as in (d,e). Numbers indicate relative GAPDH-normalized densitometric quantity. (g,h) RT-qPCR measurements of endogenous (g) circPVT1 and (h) NRAS in HCT116 cells, as in (d,e). (i) Western blot for NRAS protein levels in HCT116 cells, as in (f). (j) RT-qPCR measurements of endogenous transcript levels of let-7 targets KRAS, MYC, and HMGA2 in A549 cells upon circPVT1 knockdown. One-sample t-test of fold change against unity with Holm-Šidák correction for multiple comparisons. (k) RT-qPCR measurement of hsa-let-7a-5p in A549 cells upon circPVT1 knockdown. One-sample t-test of fold change against unity. Data are shown as fold changes (mean ± S.E.M. from at least three independent experiments) relative to the control set-up. *P < 0.05, **P < 0.01, ***P < 0.001. Full-length and replicate blots are shown in Supplementary Figure S1.