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. 2020 Sep 29;11:576903. doi: 10.3389/fimmu.2020.576903

Figure 3.

Figure 3

Hsa_circ_0060450 serves as a sponge of miR-199a-5p. (A) The predicted target sequence of miR-199a-5p, miR-133a or miR-133b in hsa_circ_0060450 and corresponding mutant target sequence for binding sites-mutant luciferase assay. (B) The construction of hsa_circ_0060450 fragment-containing psicheck2 luciferase vector. (C) A luciferase reporter assay was used to detect the luciferase activity of 293T cells co-transfected with blank psicheck2 vector or psicheck2 recombinant vector containing hsa_circ_0060450 fragment and miR-199a-5p, miR-133a, miR-133b mimics, or NC. (D) A luciferase reporter assay was used to detect the luciferase activity of 293T cells co-transfected with psicheck2 recombinant vector containing mutant hsa_circ_0060450 fragment and miR-199a-5p or NC. (E) STAT1 and STAT3 protein phosphorylation assessment and quantification analyses at 15 and 30 min of IFN-I stimulation after treatment with miR-199a-5p mimics. (F) RT-qPCR analyses of IFIT1, IFIH1, CXCL10, and iNOS under IFN-I stimulation after treatment with miR-199a-5p mimics. NC, negative control. circ_0450-v, psicheck2 recombinant vector containing WT hsa_circ_0060450 fragment. circ_0450-miR199a-mut-v, psicheck2 recombinant vector containing miR-199a-5p binding site-mutant hsa_circ_0060450 fragment. Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significance.