Figure 5.

AR is a direct target of miR-224-5p

(A) The two-stage predicted wild-type (WT) and corresponding mutant (Mut) binding sites of the AR 3′ UTR with miR-224-5p. The vertical lines data indicate the binding sites between miR-224-5p and the AR WT 3′ UTR, and arrows indicate mutagenic nucleotides of the AR Mut 3′ UTR. (B and C) Relative firefly luciferase expression in HEK293T cells co-transfected with pGL3-AR-WT-3′ UTR-1/Mut-3′ UTR-1 (B) or pGL3-AR-WT-3′ UTR-2/Mut-3′ UTR-2 (C), including pRL and NC/miR-224-5p mimic. Renilla luciferase expression was used for as an internal control. (D–F) qRT-PCR analysis (D), western blot analysis (E), and quantification (F) of AR mRNA or protein expression in H1299 and A549 cells stably transfected with pLenti/pLenti-miR-224 or transiently transfected with NC/miR-224-5p inhibitor. (G) qRT-PCR analysis of AR mRNA expression in BEAS-2B, H1299, and A549 cells fed with EXO-pLenti or EXO-pLenti-miR-224. (H) qRT-PCR analysis of AR mRNA expression and relationship with tumor size, sex, and pathological stage in 83 pairs of NSCLC patient tumor and corresponding para-carcinoma tissues. (I) qRT-PCR analysis of AR mRNA expression in human lung epithelial cell line BEAS-2B, and in NSCLC cell lines 95-D, PC-9, H1975, H1299, and A549. 18S rRNA and GAPDH were used as internal controls for qRT-PCR and western blot analysis, respectively. (J) Kaplan-Meier curve of overall survival of 1,926 lung cancer patients from a Kaplan-Meier plotter online database. (K) The negative correlation between miR-224-5p and AR, according to the Pearson correlation coefficient. Error bars represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.