Fig 1: Identification of FOXP4 as a miR-3180-3p target gene. (a) The wild-type and mutated binding site between miR-3180-3p and FOXP4. (b, c) A549 and H460 cell lines were cotransfected with miR-NC and miR-3180-3p with a WT and MUT 3'UTR. Luciferase activity was measured after 24 hours cotransfection. A549: , miR-NC; , miR-3180-3p. H460: , miR-NC; , miR-3180-3p. (d, e) A549 and H460 cells were transfected with miR-NC or miR-3180-3p mimics for 48 hours. The mRNA and protein levels of FOXP4 were analyzed using qRT-PCR and western blotting , A549; , H460; , miR-NC; , miR-3180-3p. (f, g) The mRNA and protein levels of FOXP4 were analyzed using qRT-PCR and western blotting in HBE, A549 and H460 cells. (h) FOXP4 mRNA expression levels were extracted from The Cancer Genome Atlas (TCGA). *P < 0.05, **P < 0.01, and n = 3.
Fig 2: Knockdown of FOXP4 inhibits proliferation, migration and invasion in NSCLC cells. (a) The protein levels of FOXP4 in A549 and H460 cells were analyzed by western blotting after transfection with siRNA targeting FOXP4 , si-NC; , si-FOXP4-1; , si-FOXP4-2. (b) A CCK-8 assay was performed in A549 and H460 cells after suppression of FOXP4 using siRNAs. A549: , si-NC; , si-FOXP4-1; , si-FOXP4-2. H460: , si-NC; , si-FOXP4-1; , si-FOXP4-2. (c, d) The migratory and invasive ability of A549 and H460 cells were measured using a transwell assay after knockdown of FOXP4 by siRNAs. A549: , si-NC; , si-FOXP4-1; , si-FOXP4-2. H460: , si-NC; , si-FOXP4-1; , si-FOXP4-2. *P < 0.05, **P < 0.01, and n = 3.
Fig 3: Partial reversal of the inhibitory effects of miR-3180-3p on NSCLC cell phenotypes through FOXP4. A549 and H460 cells were transfected with miR-NC, miR-3180-3p, miR-3180-3p + FOXP4 or FOXP4. (a, b) Western blots were performed to detect FOXP4 protein expression levels. (c, d) A CCK-8 assay was performed. A549: , NC; , miR-3180-3p; , FOXP4; , FOXP4+miR-3180-3p. H460: , NC; , miR-3180-3p; , FOXP4; , FOXP4+miR-3180-3p. (e, f) The migratory and invasive ability of A549 and H460 cells were measured using a transwell assay. A549: , miR-NC; , miR-3180-3p; , FOXP4+miR-3180-3p; , FOXP4. H460: , miR-NC; , miR-3180-3p; , FOXP4+miR-3180-3p; , FOXP4. *P < 0.05, **P < 0.01, and n = 3.
Fig 4: FOXP4 is the target of miR-3184-5p in PCa.a The binding sequence of miR-3184-5p to FOXP4 3'UTR. b Luciferase activity of reporters containing wild type FOXP4 (FOXP4-WT) or mutant type FOXP4 (FOXP4-MUT) in cells transfected with miR-3184-5p mimics. c MiR-3184-5p expression in PCa samples and normal samples. d Expression association between miR-3184-5p and FOXP4-AS1 or FOXP4 in PCa samples. e Kaplan–Meier analysis of patients with PCa with high or low expression level of miR-3184-5p. f MiR-3184-5p expression in PCa cell lines and normal cell line. g, h. The effect of miR-3184-5p inhibitor or mimics on cell proliferation or apoptosis. **P < 0.01, ***P < 0.001
Fig 5: FOXP4-AS1 acted as the molecular sponge of miR-3184-5p.a, b FOXP4-AS1 was predominantly located in the cytoplasm of PCa cells. Scale bar = 200 µm. c Ago 2-RIP revealed that both FOXP4-AS1 and FOXP4 were enriched in Ago 2 containing beads. d Five miRNAs that had complementary base paring with both FOXP4-AS1 and FOXP4 were predicted from starBase and DIANA. e Enrichment of five miRNAs in MS2 with or without FOXP4-AS1. f miR-3184-5p and miR-423-5p were detected in cells transfected with FOXP4-AS1 expression vector or sh-FOXP4-AS1#1. g The predicted binding sequence between FOXP4-AS1 and miR-3184-5p. h The luciferase reporter assay was conducted in cells transfected with miR-3184-5p mimics or miR-NC to examine the luciferase activity of FOXP4-AS1-WT and FOXP4-AS1-MUT. *P < 0.05, **P < 0.01
Supplier Page from Abcam for Anti-FOXP4 antibody