Fig. 1

circPSD3 is downregulated in PVTT tissues and is associated with the prognosis of patients with HCC. (A) Clustered heatmap of differentially expressed circRNAs in five paired PVTT and HCC tissues. Rows represent circRNAs while columns represent tissues. (B) Volcano plot of dysregulated circRNAs in paired PVTT and HCC tissues. (C) qRT-PCR validation of the top four up and downregulated circRNAs in 19 paired PVTT tissues. (D) Relative expression of circPSD3 in 48 paired HCC and non-cancerous tissues. (E) Differences in circPSD3 expression between HCC tissues with and without MVI. (F and G) Kaplan–Meier curves showing the correlation between circPSD3 expression and recurrence-free survival and overall survival. Survival curves were compared using the log-rank test. (H) Schematic showing that exons 13 and 14 of human PSD3 circularise to produce circPSD3. The back-splice junction site was confirmed by Sanger sequencing. (I) The circular structure of circPSD3 was confirmed in HCC cell lines using convergent and divergent primers. GAPDH served as the reference gene. (J) Circular and linear isoform stability was assessed using RNase R treatment. (K) Relative RNA levels of circPSD3 and linear PSD3 in HCC cell lines treated with actinomycin D. (L) The subcellular localisation of circPSD3 in HCC cell lines using nuclear/cytoplasmic RNA fractionation. U6 and β-actin served as the nuclear and cytoplasmic controls, respectively. (M) FISH visualisation of the subcellular localization of circPSD3 in HCC cell lines. U6 and 18 S ribosomal RNA served as the nuclear and cytoplasmic controls, respectively. Scale bar = 20 μm. Data are shown as the mean ± SD. Statistical analyses were performed using unpaired Student’s t-tests (*p < 0.05; **p < 0.01; ***p < 0.001). circPSD3, circRNA pleckstrin and Sect. 7 domain containing 3; FISH, fluorescence in situ hybridisation; HCC, hepatocellular carcinoma; MVI, microvascular invasion; PVTT, portal vein tumor thrombosis; qRT-PCR, quantitative real-time PCR