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. 2023 Sep 13;42:239. doi: 10.1186/s13046-023-02813-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Circ-RAPGEF5 identification and validation in ICC cells and tissues. A Heatmap of the top 50 upregulated and downregulated expression circular RNAs between ICC tissues and adjacent normal tissues according to the bulk RNA sequencing of 19 patients. B Venn diagram illustrating the overlap of top 20 upregulated expression circular RNAs in our data and Chen et al.’ s data, only Circ-RAPGEF5 was identified. C Volcano plot of upregulated expressed circular RNAs from 19 paired samples. D Relative Circ-RPAGEF5 expression in 19 ICC tumor tissues and matched adjacent normal tissues. E qRT-PCR analysis of the relative expression levels of Circ-RAPGEF5 in a panel of four human ICC cell lines (HUCCT1, 9810, RBE, and CCLP1) and a normal biliary epithelial cell line, HIBEC. F The schematic illustration showed the back-splicing of Circ-RAPGEF5, and Sanger sequencing validated the splicing site. G Circular formation of Circ-RAPGEF5 was confirmed through PCR and agarose gel electrophoresis using divergent and convergent primers in both gDNA and cDNA samples of RBE. GAPDH was used as a negative control. H-I qRT-PCR analysis of Circ-RAPGEF5 and linear RAPGEF5 expression levels after Actinomycin D treatment in RBE and CCLP1. J-K qRT-PCR analysis of Circ-RAPGEF5 compared to linear GAPDH and RAPGEF5 expression levels after RNase R treatment in RBE and CCLP1. L-N Subcellular fractionation and FISH assays indicated that Circ-RAPGEF5 was localized in both the cytoplasm and nucleus of RBE and CCLP1. All data are presented as the means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001