Fig 2: ADAR1 is highly expressed in CRPCa. (A) Representative images and (B) quantification of immunohistochemical staining for ADAR1 in BPH and PCa tissues. (C and D) Western blot analysis of ADAR1 protein expression in different tissue types. (E) Reverse transcription-quantitative PCR and (F and G) western blot analysis of the mRNA and protein expression levels of ADAR1 in different cell lines. *P<0.05 and **P<0.01. ADAR1, double-stranded RNA-specific adenosine deaminase; BPH, benign prostatic hyperplasia; PCa, prostate cancer; CRPCa, castration-resistant PCa.

Fig 3: ADAR1-knockdown induces the apoptosis of DU145 and PC3 cells by promoting the phosphorylation of H2AX. Lysates from transfected (A) DU145 and (B) PC3 cells were analyzed via western blotting to detect the expression levels of apoptotic markers (cleaved caspase-3, caspase-3, cleaved PARP and PARP), and the DNA double-strand-break markers, p-H2AX and H2AX. Data are representative of three independently performed experiments. Protein expression levels of p-H2AX, H2AX, cleaved caspase-3, caspase-3, cleaved PARP and PARP in (C) DU145 and (D) PC3 cells. Statistical results are presented as the mean ± SD of five independent experiments. *P<0.05 and **P<0.01. ADAR1, double-stranded RNA-specific adenosine deaminase; si (RNA), small interfering; NC, negative control; p-, phosphorylated; H2AX, H2A.X variant histone.

Fig 4: ADAR1 regulated circular RNA-circCDK17. (A–B) High-throughput whole transcriptome sequencing showing the RNA changes upon silencing of ADAR1 (A) and the proportion of different types of RNA regulated by ADAR1 (B). (C) Volcanic map of differentially expressed circRNA after silencing of ADAR1, and screening of candidate circRNAs regulated by ADAR1. (D) qPCR analysis showing the expression of circCDK17, circAPBB2, circCDC14B and circTBCB upon silencing of ADAR1, n = 6. (E) Specific primers were designed for circCDK17 cycle-site for qPCR verification, and Sanger sequencing was performed to verify the accuracy of cycle-site. (F) RNA pull-down experiment showing that circCDK17 bound directly to ADAR1, n = 3. (G) Fluorescence in situ hybridization (FISH) showing the colocalization of ADAR1 and circCDK17. (H) qPCR analysis showing the expression of circCDK17 in serum of PH patients (CON = 15, PH = 10). CON, Control; PH, Pulmonary hypertension; NOR, normoxia; HYP, hypoxia; NC, negative control; SI, siRNA of ADAR1; Statistical analysis was performed with two-way ANOVA followed by Dunnett's test or the Student's t-test; All values are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig 5: ADAR1 overexpression induces AZIN1 RNA mutation. (A) Confirmation of flag-tagged ADAR1 p110 expression in Fa2N4 cells. Representative results of two independent experiments are shown. (B) AZIN1 RNA mutation in cells overexpressing flag-ADAR1 p110 was detected by ddPCR. A representative image of droplet counts containing mutant and wild-type AZIN1 cDNA amplicons from three independent experiments. (C) No mutation was detected at the AZIN1 locus in the genomic DNA of Fa2N4 control or ADAR1-expressing cells by ddPCR. Amplicons of AZIN1 RNA mutation from Fa2N4-ADAR1-expressing cells were used as positive controls. Representative results from three independent experiments are shown. ADAR, adenosine deaminase acting on RNA-1; AZIN, adenosine to inosine RNA editing of antizyme inhibitor 1; dd, digital droplet; NC, negative control.