Fig. 1: CircEZH2 is highly expressed in GBM and negatively correlated with the NK cell activation signature.

a Volcano plot of differentially expressed circRNAs in 12 paired human GBM samples. b Box plot of the length distribution of circORFs in tumour-upregulated circRNAs and the corresponding mORFs of the source genes. Data are presented as boxes containing the median (centre line), the first and third quartiles (box limits). The whiskers indicate the maxima and minima. c Expression distribution (x-axis) and length distribution (y-axis) of circORFs in tumour-upregulated circRNAs. The red dots represent the 10 candidate circRNAs. d Heatmap of the fold change values of the 10 circRNAs in (c). e Pathways negatively correlated with circEZH2 in GSEA. f Correlation analysis between circEZH2 and the functional NK cell-associated marker gene set (27 genes) in GSEA. The heatmap on the right shows the Pearson correlation coefficient between each marker and circEZH2. The vertical bars in the figure indicate the ranks and enrichment scores of the 27 genes as determined by GSEA. Ten core enriched genes are marked in the plot. g Illustration of the annotated genomic region of EZH2, the putative different RNA splicing forms. Convergent and divergent primers were designed to amplify the linear- or back-spliced products. h Left, PCR analysis using the indicated primers. Right, subsequent Sanger sequencing identified the junction sequence of circEZH2 in MES28 cells. i Left, illustration of the circEZH2 junction-specific FISH probe and circEZH2 shRNA target site. Right, FISH was performed to identify the subcellular localization of circEZH2. CircEZH2 OV plasmids and shRNA were used independently or in combination to verify the specificity of these probes. Scale bar, 10 μm. j Relative circEZH2 RNA levels in NHAs and GSC lines. 456 vs NHA, P = 0.0389; 4121 vs NHA, P = 0.0056; MES28 vs NHA, P = 0.0056; GSC23 vs NHA, P = 0.0005. k Fold change in circEZH2 expression in tumour specimens and paired adjacent brain tissues in a cohort of high-grade glioma patients (n = 63). l Relative circEZH2 expression levels in the same cohort (n = 63). Two-sided paired t test, P = 0.0013. The data in (h)–(l) are pooled  from three independent experiments. The data are presented as the mean ± SD. Unpaired two-tailed Student’s t test was used to determine the significance of differences between the indicated groups where applicable. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.