Fig. 1

Identification and characteristics of upregulated circPDHK1 in ccRCC. A The workflow for circRNA screening in 4 paired ccRCC tissues as indicated. B Cluster heatmap showed the differentially expressed 233 circRNAs in the ME blue module with WGCNA generated from 4 ccRCC tissues and paired adjacent normal tissues. The red and blue strips indicate up-regulated and down-regulated circRNAs, respectively. C Relative normalized signal detected by circRNA probe in microarray analysis of the 9 candidate circRNAs that intersected with the 233 circRNAs in the GSE100186 data. D RT-qPCR analysis of circPDHK1 expression in 148 paired ccRCC samples and normal adjacent tissues. E Comparison of circPDHK1 expression between patients with WHO/ISUP stage III–IV (n = 51) and those with WHO/ISUP stage I–II (n = 97), detected by RT-qPCR. F Comparison of circPDHK1 expression between patients with tumor T stage T1a-T1b (n = 103) and those with tumor T stage T2a-T4 (n = 45), detected by RT-qPCR. G Association analyses of circPDHK1 expression and distant tumor metastasis. H Relationships between circPDHK1 expression and VHL mutations in ccRCC paired tissues with genetic test reports (n = 106). I Relationships between circPDHK1 expression and Ki-67 in ccRCC paired tissues (n = 112). J Illustration of circPDHK1 formation by splicing exons 2 and 8 from the PDHK1 parental gene as identified in CircBase (http://circrna.org). Specific divergent and convergent PCR primers to detect circPDHK1 and linear PDHK1 are indicated, respectively. K PCR products of circPDHK1 in cDNA and gDNA amplified using convergent or divergent primers in Caki-1 and 786-O. L Schematic illustration of circPDHK1 conformation. The exon 2–8 of PDHK1 mRNA formed PDHK1 through back splicing. As shown, Sanger sequencing was used to verify the back-splice junction site of circPDHK1. M Reverse transcription using oligo dT and random priming to identify circPDHK1 loop characteristics by RT-qPCR. N CircPDHK1 and linearPDHK1 mRNA stability was assessed using RNase R treatment. O RT-qPCR analysis of the abundance of circPDHK1 and PDHK1 linear mRNA in ccRCC cells treated with actinomycin D at the indicated time points. P RT-qPCR analysis of circPDHK1 location in the nucleus or cytoplasm in Caki-1 and 786-O cells. GADPH served as a marker of cytoplasmic location, while U6 served as a marker of nuclear location. Q Fluorescence in situ hybridization (FISH) was utilized to detect circPDHK1 localization in Caki-1 and 786-O cell lines. Bars = 20 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance