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. 2020 Jun 15;28(11):2473–2487. doi: 10.1016/j.ymthe.2020.06.013

Figure 4.

Figure 4

lncAMPC Upregulates the Expression of LIF by Competitively Binding and Then Inhibiting the Activity of miR-637

(A) Quantitative real-time PCR analysis of miR-637 and LIF expressions in C4-2 and PC-3 transfected with NCsi, silncAMPC-1, or silncAMPC-2 (n = 3). (B) Quantitative real-time PCR analysis of lncAMPC and LIF expressions in C4-2 and PC-3 transfected with miR-637 mimics or control mimics (n = 3). (C) Western blot analysis of LIF expression in C4-2 and PC-3 transfected with silncAMPC-2 and/or miR-637 inhibitor (n = 3). (D) MS2-based RIP assay with anti-GFP antibody followed by miRNA quantitative real-time PCR to detect the binding ability of lncAMPC to miR-637 (n = 3). (E) Anti-Ago2 RIP assay followed by quantitative real-time PCR to detect the binding ability of Ago2 to lncAMPC (n = 3). (F and G) RIP assay of the enrichment of Ago2 on lncAMPC and LIF transcript relative to immunoglobulin G (IgG) in PC-3 cells transfected with lncAMPC-OV or NC-OV (F), and with NCsi or silncAMPC-2 (G) (n = 3). (H) Schematic outline of predicted binding site for miR-637 on lncAMPC; the red nucleotides (binding site) were mutated in the mutant constructs. (I) Luciferase activity of psiCHECK2-lncAMPCwt and psiCHECK2-lncAMPCmt upon transfection of miR-637 mimics in 293T cells (n = 3). Data are presented as the ratio of Renilla luciferase activity to Firefly luciferase activity. Results are presented as mean ± SD; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.