Figure 6
Identification of RIG-I as potential receptor for miR-136.
(A) Expression of IL-6 and IFN-β was measured in A549 cells following cotransfection with the indicated RIG-I expression plasmids and either miR-136 or NC. (B) IFN-β mRNA expression levels were measured in 293T cells following the indicated treatments. (C) Validation of Flag-tagged RIG-I expression in 293T and A549 cells by western blotting. (D) Expression of RIG-I mRNA in A549 cells was determined following miR-136 transfection (60 nM, final concentration). (E) RIG-I protein levels in cells transfected with miR-136, miR-145, or NC were analysed by western blotting, using an anti-RIG-I antibody. (F) IL-6, IFN-β and RIG-I mRNA expression levels after sequential 24-hour transfections, first with a siRNA targeting RIG-I (siRIG-I; 60 nM) or a scrambled siRNA and then with 60 nM miR-136. (G) A549 cells were transfected for 24 hours with the indicated concentrations of miR-145, miR-145 mixed with 2′-O-methyl modified miR-136, or modified NC. Subsequently, mRNA expression of CCL5, CXCL10 and IFN-β were measured by RT-qPCR. The results are presented as mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by one-way ANOVA with Bonferroni’s multiple comparison test (A,B,G) or Student’s t test (D,F). Emp, empty vector; scr, scrambled siRNA; 2′M, 2′-O-methyl modified. The blots were run under the same experimental conditions. Full-length blots/gels are presented in Supplementary Figures 6, 7 and 8.
