Figure 4.

MiR-30c-5p targeted the 3′ UTRs of SOCS1. (A) Schematic diagram of the left panel is the predicted target sites of miR-30c-5p in the SOCS1 3′ UTRs of seven representative mammals. The predicted target sites and mutated target sites of miR-30c-5p are underlined and mutated as indicated (right panel). (B) Results of the luciferase assay. Vero E6 cells were co-transfected with SOCS1 wild-type or mutant luciferase vectors (500 ng) and 160 nM of miR-30c-5p mimics or NC mimics, miR-30c-5p inhibitor, or NC inhibitor, and the luciferase activity was analyzed at 24 h after transfection. FL, firefly luciferase; RL, Renilla luciferase. (C) The suppression of SOCS1 protein levels by miR-30c-5p under PEDV-uninfected and -infected conditions. Vero E6 cells were transfected as described in the legend for panel B for 24 h, followed by infection with PEDV (MOI=0.1) or mock infection with DMEM, and the samples were collected at 36 h for Western blotting of SOCS1 or β-actin. Quantifications were normalized to those of uninfected NC. (D,E) MiR-30c-5p increased the anti-PEDV activity of IFN-λ. After transfection with miR-30c-5p mimics or inhibitor for 24 h, cells were pretreated with IFN-λ or DMEM for 12 h and then infected with PEDV (MOI = 0.1) and harvested at 36 hpi for viral RNA quantification and TCID₅₀. (F) The SOCS1 expression levels in Vero E6 cells were measured by RT-qPCR at 36 hpi at different MOIs. P values represent the difference from the mock-infected control for time kinetics, the SOCS1, and miR-30c-5p levels. Error bars, mean ± SEM. (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, and NS, not significant.