Fig. 1: Downregulation and characterization of circSPIRE1 in metastatic RCC.

A Schematic illustration of the development of the metastatic RCC PDX model, PDX LM. B Increased tumor metastasis formed in lungs of PDX LM model compared to the original PDX model. Left, representative lung and representative HE staining of lung metastatic lesions, scale bar, 100 µm. Right, the number of metastatic nodules formed in the lungs of mice for each group (n = 6 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test. C Increased microvessel density of the PDX LM model compared to the original PDX model. Left, representative tumor stained with CD34 anti-body. Right, microvessel density for each group (n = 5 mice/group). Data represent mean ± S.D.; dot plot reflects data points from independent experiments. The P values were determined by Student’s t test. D Heatmap of high-throughput sequencing between PDX LM cells and original PDX cells. E Volcano plot of circRNAs comparing five no clinically metastasis cases and five metastasis cases. F qRT-PCR analysis of four circRNAs expression in 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; The P values were determined by Student’s t test. G Left, flowchart illustrating the screening criteria of potential metastasis related circRNAs. Right, Kaplan−Meier analysis of DFS in RCC patients with low versus high expression of circSPIRE1. The P value was determined by a Log-rank test. H Existence of circSPIRE1 confirmed by RT-PCR and gel electrophoresis using convergent and divergent primers. CircSPIRE1 can only be amplified in cDNA. I Northern blotting of circSPIRE1 and SPIRE1 transcripts by hybridization with exon 4 (left) and exon 6−exon 4 junction (right) probes in the presence or absence of RNase R treatment. J RNA fluorescence in situ hybridization for circSPIRE1. Scale bar, 10 µm. K Cytoplasmic and nuclear RNA fractionation experiment showing that circSPIRE1 mainly localized in the cytoplasm. GAPDH and U3 as positive controls in the cytoplasm and nucleus, respectively.