Figure 7.

CSF2RA and CD43 are downregulated by miR-7083-5p as candidate targets. (A) Functional categorization of 33 predicted targets of miR-7083-5p. Functional enrichment analysis were performed using DAVID software. (B) Normalized expression levels of the four immune/inflammatory response-related genes from the Human Protein Atlas. (C) Experimental schemes of reporter assays using pmirGLO dual-luciferase miRNA target expression vector inserted with CSF2RA- or CD43-3′-UTR and a control (Ctrl) vector. (D) Firefly luciferase activity normalized to Renilla luciferase activity (FF/R) of HEK293T cells transfected with the pmirGLO luciferase reporter in the absence or presence of miR-7083-5p or a scrambled control. (E) Flow cytometric analysis of CSF2RA and CD43 expression levels in M2 macrophages (M2) after transfection with miR-7083-5p (M2mi). M1 macrophages (M1) were used as control. (F) qRT-PCR analysis of relative mRNA levels of csf2ra and cd43 in M2 macrophages after transfection with miR-7083-5p. (G, H) qRT-PCR analysis of the relative mRNA levels of csf2ra and cd43 in 4T1 (G) and LLC (H) tumor tissues after treatments with 5-aza-dC (referred as A) and TSA (referred as T), alone or in combination (A+T). Data have been presented as the mean ± S.D. of three separate experiments performed in five replicates. *, P<0.05; **, P<0.01; ***, P<0.001; n.s., not significant, as assessed using two-way ANOVA followed by Bonferroni multiple comparisons post-hoc test (B) and one-way ANOVA followed by Tukey’s multiple comparison post-hoc test (C–F).