Fig. 1: Validation and expression of circZNF566 in HCC tissues and cells.

a CircRNA microarray comprising five pairs of HCC tissues and matched normal liver tissues. b Schematic illustration of the formation of circZNF566 via circularization of exons in the ZNF566 gene. The head-to-tail splicing of circZNF566 was confirmed by Sanger sequencing. c CircZNF566 expression in HCC and normal liver cell lines was detected by qRT-PCR. d The expression of circZNF566 and ZNF566 mRNA in both Huh7 and LM3 cells was validated by qRT-PCR in the presence or absence of RNase R. e qRT-PCR analysis of circZNF566 and ZNF566 mRNA after treatment with actinomycin D at the indicated time points in Huh7 and LM3 cells. f CircZNF566 was predominantly localized to the cytoplasm as indicated by a nuclear-cytoplasmic fractionation assay. g Analysis of the relatively differential expression levels of circZNF566 between 57 pairs of fresh frozen HCC and matched normal tissues. h The level of circZNF566 was obviously higher in stage M1 HCC tissues than in stage M0 HCC tissues and in UICC stage III–IV tumors than in UICC stage I–II tumors. i Kaplan–Meier survival analysis showed that HCC patients with high circZNF566 expression had a lower OS (p = 0.018) and DFS (p = 0.007) than those with low circZNF566 expression. All data are from three independent experiments and are presented as the means ± SEM or representative of three independent experiments with similar results (*p < 0.05, **p < 0.01, ***p < 0.001).