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. 2022 Jul 25;54(7):1024–1037. doi: 10.1038/s12276-022-00806-z

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a The expression of certain ROCK1-derived circRNAs in osteosarcoma tissue specimens and paired paratumor tissue specimens was determined by an RT–qPCR assay. b Representative images of circROCK1-E3/E4 expression in osteosarcoma and paratumor tissues as displayed by a FISH assay. c The expression of circROCK1 in 50 osteosarcoma and paired paratumor tissue specimens was detected by RT–qPCR assay. ^****p < 0.0001. d CircROCK1-E3/E4 was downregulated in osteosarcoma cell lines, as confirmed by an RT–qPCR assay. ^****p < 0.0001. e CircROCK1-E3/E4 is circularized from exons 3 and 4 in ROCK1, as demonstrated by a schematic illustration. f CircROCK1-E3/E4 was amplified by divergent primers in cDNA but not gDNA by using GAPDH as a negative control, as validated by an RT–qPCR assay. g Compared with oligo (dT)₁₈ primers, the expression of circROCK1-E3/E4 was significantly decreased when random hexamer primers were applied. ^n.sp > 0.05 and ^**p < 0.01. h Compared with linear ROCK1 mRNA, the expression changes of circROCK1-E3/E4 were unremarkable in the presence of RNase R. ^n.sp > 0.05 and ^**p < 0.01. i The RNA levels of circROCK1-E3/E4 and ROCK1 mRNA after 2 μg/ml actinomycin D treatment were quantified at different time points by qRT–PCR assays. ^**p < 0.01. j CircROCK1-E3/E4 was mainly localized in the cytoplasm of U2OS and HOS cells. k CircROCK1-E3/E4 is frequently downregulated in patients with lymph node metastasis (N1 and N2). ^*p < 0.05, ^****p < 0.0001. l CircROCK1-E3/E4 is downregulated in patients with distant metastasis (M1 stage). ^****p < 0.0001. m ROC curve analysis of the area under the curve (AUC) and 95% confidence interval (CI) of circROCK1-E3/E4 in patients with osteosarcoma. n A low expression level of circROCK1-E3/E4 was correlated with shorter survival rates in patients with osteosarcoma as determined by Kaplan–Meier analysis. All data are presented as the mean ± SD from three independent experiments.