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. 2017 May 4;8(5):e2764. doi: 10.1038/cddis.2017.145

Figure 3.

Figure 3

IRF2 is a direct target of miR-18a-5p. (a) IRF2 WT 3'-UTR contains predicted miR-18a-5p binding sites. The figure shows alignment of miR-18a-5p with the IRF2 WT 3'-UTR, with arrows indicating the mutagenesis nucleotides. (b) Dual luciferase reporter assay. HEK293T cells were co-transfected with luciferase reporter constructs containing the pGL3-IRF2 WT 3'-UTR (IRF2 WT) and the pGL3-IRF2 mut 3'-UTR (IRF2 mut) with miR-18a-5p mimic or NC mimic. Relative firefly luciferase expression is displayed, normalized to Renilla luciferase expression, n=3 independent experiments. (c) Immunoblotting analysis of IRF2 levels in H23, H1299 and A549 cells transfected with NC mimic/miR-18a-5p mimic or NC inhibitor/miR-18a-5p inhibitor for 48 h. (d) The relative expression of IRF2 mRNA from qRT-PCR of corresponding non-tumour tissues (NT) and tumour tissues (T). 18 S was used for normalization. (e-g) Expression of IRF2 related to tumour size, sex and pathological stage. (h) The relative expression of IRF2 mRNA in lung cancer cell lines or a pulmonary epithelial cell line, n=3 independent experiments. (i) The effect of IRF2 expression levels on the overall survival in 1926 lung cancer patients (Kaplan–Meier Plotter online database) was analysed and Kaplan–Meier plots were generated using a Kaplan–Meier plotter (http://www.kmplot.com). (j) MiR-18a-5p had a negative correlation with IRF2 according to Pearson correlation coefficient. Error bars represent the mean±S.E.M. * P<0.05, **P<0.01, ***P<0.001