High-throughput circular RNA sequencing reveals tumor-specific high expression of hsa_circ_0001900 in Wilms tumor in association with poor prognosis

doi:10.12122/j.issn.1673-4254.2025.11.19

Abstract

Abstract:

Objective To explore the expression profile of circular RNAs (circRNAs) and their potential roles in prognosis and progression of Wilms' tumor (WT). Methods Four pairs of WT and adjacent tissues were collected for high-throughput circRNA sequencing to identify the differentially expressed circular RNAs. RT-qPCR was used to verify the expression levels of the top 6 candidate circRNAs in the clinical samples. hsa_circ_0001900 was selected for analysis of its correlation with clinicopathological features and prognosis in 34 patients with WT. Sanger sequencing and RNase R digestion experiments were used to verify the cycling site and structural stability of hsa_circ_0001900 molecule. Results A total of 23 978 circular RNA molecules were identified in WT tissues by high-throughput circular RNA sequencing, and among them 614 were differentially expressed in WT. hsa_circ_0001900 showed the highest expression level among the differentially expressed circRNAs, which was consistent with the findings in clinical tumor samples and the sequencing results. Correlation analysis showed that hsa_circ_0001900 expression level was positively correlated with WT volume, and the children with high hsa_circ_0001900 expression had a lowered recurrence-free survival rate. The results of Sanger sequencing verified the circular splice site sequence of the molecule, and Rnase R digestion assay confirmed its stable covalent structure. Conclusion This study presents a comprehensive expression profile of circular RNAs in WT, and the expression level of hsa_circ_0001900 is related to the size of WT and the patients' prognosis, suggesting its possible role as a key driving gene in WT progression.

Key words: nephroblastoma, Wilms tumor, circular RNA, hsa_circ_0001900

Zhiqiang GAO, Jie LIN, Peng HONG, Zaihong HU, Kongkong CUI, Yu WANG, Junjun DONG, Qinlin SHI, Xiaomao TIAN, Guanghui WEI. High-throughput circular RNA sequencing reveals tumor-specific high expression of hsa_circ_0001900 in Wilms tumor in association with poor prognosis[J]. Journal of Southern Medical University, 2025, 45(11): 2466-2474.

Figures/Tables 9

Tab.1 Quality control for sequencing results

Samples	Output of data (Gb)	Q30 (%)
Tumor tissue-1	15.060	88.84
Tumor tissue-2	13.910	88.83
Tumor tissue-3	14.581	89.52
Tumor tissue-4	13.873	89.72
Adjacent control-1	13.325	90.70
Adjacent control-2	14.272	90.45
Adjacent control-3	15.289	90.44
Adjacent control-4	11.826	90.67

Tab.2 Primer sequences for q-PCR

Genes	Pre-primer (5'→3')	Post-primer (3'→5')
hsa_circ_0001900	CGTTCAGTGCCTCGAAAGAAC	CTGGTCCCCTTTCAGGATGAG
hsa_circ_0004425	AAGTGTGGACCCTGCAAATA	CCATTTGATCCACAGACAGGA
hsa_circ_0005230	CTCTTTGTTTTGCACACTAGGGA	ACCAGGTGAGCAGTCAAGAA
hsa_circ_0075828	GGAAGTGGCTAATGGATCTG	ATGGAGAACAGCCATCCATG
hsa_circ_0008122	TGTCTCAAAGCCAGACCTGAT	ATTCTGTTGTTCGGTGTCCAG
hsa_circ_0006151	GGCACCAGCATAGCCAGTAGT	GATGACCAGGCGCTTTTCACA
GAPDH	CCTTCCTGGGCATGGAGTC	TGATCTTCATTGTGCTGGGTG

Fig.1 Identification and annotation of circRNAs A: Schematic diagram of Wilms tumor (WT) circular RNA sequencing analysis. B: Circos showing the full view of the circRNAs, with the outer circles representing chromosomes, the inner circles representing samples, and the red part of each sample representing "spliced" readings of the circRNAs. C: Venn diagram of circRNA sequencing data annotated with circBase database.

Fig.2 Classification and differential expression patterns of circRNAs. A: Circular RNA classification. B: Principal component analysis (PCA). C: Volcano plot of the differentially expressed circRNAs. Red dots indicate upregulation and blue dots indicate downregulation. The horizontal dashed line represents the truncated P value of 0.05, and the vertical dashed line represents the 2-fold logFC value.

Fig.3 Validation of tumor-specific expression pattern of hsa_circ_0001900 in clinical samples of WT A: Ranking map of circRNA expression abundance. B: Expression validation of the top 6 circRNAs in clinical samples. C: ROC diagnostic curve of hsa_circ_0001900. *P<0.05, **P<0.01 vs Tumor group.

Tab.3 Characteristics of circRNA molecules for expression validation in clinical samples

circRNA	Chromosomal loci	Direction of ring formation	Genome length	Splice sequence length	Gene ID	Gene of parent	Trend of expression
hsa_circ_0008122	chr19:23134080-23136043	+	1964	223	ENSG00000183850	ZNF730	up
hsa_circ_0001900	chr9:135881633-135883078	-	1446	425	ENSG00000130559	CAMSAP1	up
hsa_circ_0006151	chr10:68468123-68470163	-	2041	367	ENSG00000138346	DNA2	up
hsa_circ_0004425	chr9:100498765-100516771	+	18007	364	ENSG00000241697	TMEFF1	up
hsa_circ_0075828	Chr6:22020339-22020542	+	204	204	ENSG00000272168	CASC15	up
hsa_circ_0005230	Chr1:172140480-172144437	-	3958	3958	ENSG00000230630	DNM3OS	up

Fig.4 Correlation analysis of hsa_circ_0001900 expression level in WT with tumor volume and prognosis. A: Expression levels of hsa_circ_0001900 in different cancers. B: Verification of differential expression of hsa_circ_0001900 in a patient cohort. C: The expression level of hsa_circ_0001900 is positively correlated with tumor volume. D: Children with high expression of hsa_circ_0001900 had shorter survival time. *P<0.05, **P<0.01 vs Tumor group.

Fig.5 Annotation and verification of circular structure of hsa_circ_0001900. A: Sanger sequencing based on PCR products that verifies the reverse splicing sequence of hsa_circ_0001900. B: Agarose gel electrophoresis verifies the RNase R enzyme tolerance of hsa_circ_0001900 from a qualitative perspective. C: qPCR verifies the structural stability of hsa_circ_0001900 from a quantitative perspective. ****P<0.0001.

References 44

[1]	Zhong Y, Du Y, Yang X, et al. Circular RNAs function as ceRNAs to regulate and control human cancer progression[J]. Mol Cancer, 2018, 17(1): 79. doi：10.1186/s12943-018-0827-8
[2]	田小毛, 向彬, 刘丰, et al. 环状RNA在儿童肾母细胞瘤中的表达谱、生物学功能和临床意义 [J]. 生物化学与生物物理进展, 2023, 50(3): 463-72.
[3]	Zhao Y, Li J, Li J, et al. The decreased circular RNA hsa_circ_0072309 promotes cell apoptosis of ischemic stroke by sponging miR-100[J]. Eur Rev Med Pharmacol Sci, 2020, 24(8): 4420-9.
[4]	Anastasiadou E, Jacob LS, Slack FJ. Non-coding RNA networks in cancer[J]. Nat Rev Cancer, 2018, 18(1): 5-18. doi：10.1038/nrc.2017.99
[5]	Hare A, Zeng M, Rehemutula A, et al. Hsa-circ_0000064 accelerates the malignant progression of gastric cancer via sponging microRNA-621[J]. Kaohsiung J Med Sci, 2021, 37(10): 841-50. doi：10.1002/kjm2.12419
[6]	Hsiao KY, Lin YC, Gupta SK, et al. Noncoding effects of circular RNA CCDC66 promote colon cancer growth and metastasis[J]. Cancer Res, 2017, 77(9): 2339-50. doi：10.1158/0008-5472.can-16-1883
[7]	Yin WB, Yan MG, Fang X, et al. Circulating circular RNA hsa_circ_0001785 acts as a diagnostic biomarker for breast cancer detection[J]. Clin Chim Acta, 2018, 487: 363-8. doi：10.1016/j.cca.2017.10.011
[8]	Qiu M, Xia W, Chen R, et al. The circular RNA circPRKCI promotes tumor growth in lung adenocarcinoma[J]. Cancer Res, 2018, 78(11): 2839-51. doi：10.1158/0008-5472.can-17-2808
[9]	Zhang Y, Jiang J, Zhang J, et al. CircDIDO1 inhibits gastric cancer progression by encoding a novel DIDO1-529aa protein and regulating PRDX2 protein stability[J]. Mol Cancer, 2021, 20(1): 101. doi：10.1186/s12943-021-01390-y
[10]	Hou L, Zhang J, Zhao F. Full-length circular RNA profiling by nanopore sequencing with CIRI-long[J]. Nat Protoc, 2023, 18(6): 1795-813. doi：10.1038/s41596-023-00815-w
[11]	López-Jiménez E, Rojas AM, Andrés-León E. RNA sequencing and prediction tools for circular RNAs analysis[J]. Adv Exp Med Biol, 2018, 1087: 17-33. doi：10.1007/978-981-13-1426-1_2
[12]	Zhang J, Zhao F. Circular RNA discovery with emerging sequencing and deep learning technologies[J]. Nat Genet, 2025, 57(5): 1089-102. doi：10.1038/s41588-025-02157-7
[13]	Li J, Sun D, Pu W, et al. Circular RNAs in cancer: biogenesis, function, and clinical significance[J]. Trends Cancer, 2020, 6(4): 319-36. doi：10.1016/j.trecan.2020.01.012
[14]	Zhang J, Quan Y, Su X, et al. Circ-PRMT5 stimulates the proliferative ability in Wilms' tumor through the miR-7-5p/KLF4 axis[J]. Cell Mol Biol: Noisy-le-grand, 2023, 69(8): 232-6. doi：10.14715/cmb/2023.69.8.36
[15]	田小毛, 石秦林, 陆鹏, 等. 生物标志物对Wilms瘤预后判断及治疗指导价值的研究进展 [J]. 现代医药卫生, 2020, 36(6): 855-9.
[16]	Xiang B, Chen ML, Gao ZQ, et al. CCNB1 is a novel prognostic biomarker and promotes proliferation, migration and invasion in Wilms tumor[J]. BMC Med Genomics, 2023, 16(1): 189. doi：10.1186/s12920-023-01627-3
[17]	高志强, 林洁, 洪鹏, 等. 基于高通量RNA测序分析Wilms瘤中关键基因对预后及免疫应答的影响 [J]. 南方医科大学学报, 2024, 44(4): 727-38.
[18]	Chen S, Zhou Y, Chen Y, et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor[J]. Bioinformatics, 2018, 34(17): i884-90. doi：10.1093/bioinformatics/bty560
[19]	Gao Y, Wang J, Zhao F. CIRI: an efficient and unbiased algorithm for de novo circular RNA identification[J]. Genome Biol, 2015, 16: 4. doi：10.1186/s13059-014-0571-3
[20]	Glažar P, Papavasileiou P, Rajewsky N. circBase: a database for circular RNAs[J]. RNA, 2014, 20(11): 1666-70. doi：10.1261/rna.043687.113
[21]	Krzywinski M, Schein J, Birol I, et al. Circos: an information aesthetic for comparative genomics[J]. Genome Res, 2009, 19(9): 1639-45. doi：10.1101/gr.092759.109
[22]	Robic A, Kühn C. Beyond back splicing, a still poorly explored world: non-canonical circular RNAs[J]. Genes: Basel, 2020, 11(9): E1111. doi：10.3390/genes11091111
[23]	Li Y, Zheng Q, Bao C, et al. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis[J]. Cell Res, 2015, 25(8): 981-4. doi：10.1038/cr.2015.82
[24]	Dome JS, Graf N, Geller JI, et al. Advances in wilms tumor treatment and biology: progress through international collaboration[J]. J Clin Oncol, 2015, 33(27): 2999-3007. doi：10.1200/jco.2015.62.1888
[25]	Saltzman AF, Cost NG, Romao RLP. Wilms tumor[J]. Urol Clin North Am, 2023, 50(3): 455-64. doi：10.1016/j.ucl.2023.04.008
[26]	向彬. CircRACGAP1/miR-486-3p/CTNNBIP1调控轴促进肾母细胞瘤增殖侵袭和迁移的机制研究 [D], 重庆医科大学, 2023.
[27]	Kristensen LS, Jakobsen T, Hager H, et al. The emerging roles of circRNAs in cancer and oncology[J]. Nat Rev Clin Oncol, 2022, 19(3): 188-206. doi：10.1038/s41571-021-00585-y
[28]	Kristensen LS, Andersen MS, Stagsted LVW, et al. The biogenesis, biology and characterization of circular RNAs[J]. Nat Rev Genet, 2019, 20(11): 675-91. doi：10.1038/s41576-019-0158-7
[29]	Beilerli A, Gareev I, Beylerli O, et al. Circular RNAs as biomarkers and therapeutic targets in cancer[J]. Semin Cancer Biol, 2022, 83: 242-52. doi：10.1016/j.semcancer.2020.12.026
[30]	Lasda E, Parker R. Circular RNAs: diversity of form and function[J]. RNA, 2014, 20(12): 1829-42. doi：10.1261/rna.047126.114
[31]	Zhang F, Zou HM, Li XY, et al. CircRNA_0017076 acts as a sponge for miR-185-5p in the control of epithelial-to-mesenchymal transition of tubular epithelial cells during renal interstitial fibrosis[J]. Hum Cell, 2023, 36(3): 1024-40. doi：10.1007/s13577-023-00877-8
[32]	Wang L, Zhou Y, Jiang L, et al. CircWAC induces chemotherapeutic resistance in triple-negative breast cancer by targeting miR-142, upregulating WWP1 and activating the PI3K/AKT pathway[J]. Mol Cancer, 2021, 20(1): 43. doi：10.1186/s12943-021-01332-8
[33]	Xu F, Xiao Q, Du WW, et al. CircRNA: functions, applications and prospects[J]. Biomolecules, 2024, 14(12): 1503. doi：10.3390/biom14121503
[34]	Dong J, Zeng Z, Huang Y, et al. Challenges and opportunities for circRNA identification and delivery[J]. Crit Rev Biochem Mol Biol, 2023, 58(1): 19-35. doi：10.1080/10409238.2023.2185764
[35]	Xu K, Park D, Magis AT, et al. Small molecule KRAS agonist for mutant KRAS cancer therapy[J]. Mol Cancer, 2019, 18(1): 85. doi：10.1186/s12943-019-1012-4
[36]	Casagrande GMS, Silva MO, Reis RM, et al. Liquid biopsy for lung cancer: up-to-date and perspectives for screening programs[J]. Int J Mol Sci, 2023, 24(3): 2505. doi：10.3390/ijms24032505
[37]	Gong Y, Mao J, Wu D, et al. Circ-ZEB1.33 promotes the proliferation of human HCC by sponging miR-200a-3p and upregulating CDK6[J]. Cancer Cell Int, 2018, 18: 116. doi：10.1186/s12935-018-0602-3
[38]	Chen S, Li T, Zhao Q, et al. Using circular RNA hsa_circ_0000190 as a new biomarker in the diagnosis of gastric cancer[J]. Clin Chim Acta, 2017, 466: 167-71. doi：10.1016/j.cca.2017.01.025
[39]	Huang M, He YR, Liang LC, et al. Circular RNA hsa_circ_0000745 may serve as a diagnostic marker for gastric cancer[J]. World J Gastroenterol, 2017, 23(34): 6330-8. doi：10.3748/wjg.v23.i34.6330
[40]	Chen Z, Xu W, Zhang D, et al. circCAMSAP1 promotes osteosarcoma progression and metastasis by sponging miR-145-5p and regulating FLI1 expression[J]. Mol Ther Nucleic Acids, 2021, 23: 1120-35. doi：10.1016/j.omtn.2020.12.013
[41]	Wang Y, Li X, Wang H, et al. CircCAMSAP1 promotes non-small cell lung cancer proliferation and inhibits cell apoptosis by sponging miR-1182 and regulating BIRC5[J]. Bioengineered, 2022, 13(2): 2428-39. doi：10.1080/21655979.2021.2011639
[42]	Luo Z, Lu L, Tang Q, et al. CircCAMSAP1 promotes hepatocellular carcinoma progression through miR-1294/GRAMD1A pathway[J]. J Cell Mol Med, 2021, 25(8): 3793-802. doi：10.1111/jcmm.16254
[43]	Zhou C, Liu HS, Wang FW, et al. circCAMSAP1 promotes tumor growth in colorectal cancer via the miR-328-5p/E2F1 axis[J]. Mol Ther, 2020, 28(3): 914-28. doi：10.1016/j.ymthe.2019.12.008
[44]	Wang Y, Yan Q, Mo Y, et al. Splicing factor derived circular RNA circCAMSAP1 accelerates nasopharyngeal carcinoma tumori-genesis via a SERPINH1/c-Myc positive feedback loop[J]. Mol Cancer, 2022, 21(1): 62. doi：10.1186/s12943-022-01502-2