Fig 1: Direct targeting of the 3'-UTR of KCTD20 by miR-324-5p and inhibition of its effects by silencing of NEAT1. (A) Venn diagram showing an overlap among potential miR-324-5p target genes predicted by Targetscan, miRBD and miRWalk. (B) Western blotting and RT-qPCR analysis of the expression levels of six putative miR-324-5p target genes in U251 cells transfected with Anti-Ctrl or miR-324-5p inhibitor. (C) RT-qPCR analysis of KCTD20 expression in glioma and adjacent non-tumor tissues. (D) Schematic showing the WT-KCTD20 3'-UTR sequence, the location of the miR-324-5p binding site predicted by Targetscan and the MUT-KCTD20 3'-UTR sequence used for the luciferase reporter assay. (E) Luciferase activity in U251 and LN229 cells co-transfected with a luciferase plasmid driven by the WT- or MUT-KCTD20 3'-UTR and either control (miR-NC) or miR-324-5p mimics. (F) Western blotting and RT-qPCR of KCTD20 expression in U251 and LN229 cells transfected with Anti-Ctrl, miR-324-5p inhibitor, miR-NC or miR-324-5p mimics. (G) Western blotting and RT-qPCR analysis of KCTD20 expression in U251 and LN229 cells transfected with siCtrl or siNEAT1. (H) Western blotting and RT-qPCR analysis of KCTD20 expression in U251 and LN229 cells transfected with siCtrl or siNEAT1 with or without an miR-324-5p inhibitor. (I) Spearman's rank correlation analysis of the association between NEAT1 and KCTD20 mRNA expression in 43 glioma tissues. The experiments were performed in triplicate and data are presented as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. Anti-Ctrl, miR-NC, siCtrl or as indicated. Anti-Ctrl, control sequence; ELAVL1, ELAV like RNA binding protein 1; KCTD20, potassium channel tetramerization protein domain containing 20; KLF3, Kruppel like factor 3; miR, microRNA; MUT, mutant; NC, negative control; NEAT1, nuclear paraspeckle assembly transcript 1; NIPBL, NIPBL cohesin loading factor; RT-qPCR, reverse transcription quantitative polymerase chain reaction; siCtrl, control siRNA; siNEAT1, NEAT1-specific siRNA; siRNA, small interfering RNA; 3'UTR, 3' untranslated region; WT, wild-type; ZFX, zinc finger protein X-linked.