Mutations in the splicing factor 3b subunit 1 (SF3B1) gene are frequent in myelodysplastic neoplasms (MDS). Because the splicing process is involved in the production of circular RNAs (circRNAs), we investigated the impact of SF3B1 mutations on circRNA processing. Using RNA sequencing, we measured circRNA expression in CD34+ bone marrow MDS cells. We defined circRNAs deregulated in a heterogeneous group of MDS patients and described increased circRNA formation in higher-risk MDS. We showed that the presence of SF3B1 mutations did not affect the global production of circRNAs; however, deregulation of specific circRNAs was observed. Particularly, we demonstrated that strong upregulation of circRNAs processed from the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor; this upregulation was exclusive to SF3B1-mutated patients and was not observed in those with mutations in other splicing factors or other recurrently mutated genes, or with other clinical variables. Furthermore, we focused on the most upregulated ZEB1-circRNA, hsa_circ_0000228, and, by its knockdown, we demonstrated that its expression is related to mitochondrial activity. Using microRNA analyses, we proposed miR-1248 as a direct target of hsa_circ_0000228. To conclude, we demonstrated that mutated SF3B1 leads to deregulation of ZEB1-circRNAs, potentially contributing to the defects in mitochondrial metabolism observed in SF3B1-mutated MDS.

1st Department of Medicine General University Hospital Prague Czech Republic

Department of Biochemistry Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

Department of Computer Science Czech Technical University Prague Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University Prague Czech Republic

Department of Genomics Institute of Hematology and Blood Transfusion Prague Czech Republic

Department of Immunomonitoring and Flow Cytometry Institute of Hematology and Blood Transfusion Prague Czech Republic

Department of Modern Immunotherapy Institute of Hematology and Blood Transfusion Prague Czech Republic

Department of Proteomics Institute of Hematology and Blood Transfusion Prague Czech Republic

International Clinical Research Center of St Anne's University Hospital Brno Czech Republic

Laboratory of Anemias Institute of Hematology and Blood Transfusion Prague Czech Republic

Zobrazit více v PubMed

Khoury JD, Solary E, Abla O, Akkari Y, Alaggio R, Apperley JF, et al. The 5th edition of the World Health Organization classification of haematolymphoid tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703–1719. PubMed PMC

Bernard E, Tuechler H, Greenberg PL, Hasserjian RP, Arango Ossa JE, Nannya Y, et al. Molecular international prognostic scoring system for myelodysplastic syndromes. NEJM Evid. 2022;1(7). 10.1056/EVIDoa2200008 PubMed DOI

Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014;28(2):241–247. PubMed PMC

Boultwood J, Dolatshad H, Varanasi SS, Yip BH, Pellagatti A. The role of splicing factor mutations in the pathogenesis of the myelodysplastic syndromes. Adv Biol Regul. 2014;54(1):153–161. PubMed

Ramabadran R, Wang J, Guzman A, Cullen SM, Brunetti L, Gundry M, et al. Loss of de novo DNA methyltransferase DNMT3A impacts alternative splicing in hematopoietic stem cells. Blood. 2017;130(Suppl 1):1. PubMed

Lord AM, Clement K, Schneider RK, Marie M, Chen MC, Levine RL, et al. Loss of TET2 function in myelodysplastic syndrome results in intragenic hypermethylation and alterations in mRNA splicing. Blood. 2014;124(21):775.

Chen L, Tovar‐Corona JM, Urrutia AO. Increased levels of noisy splicing in cancers, but not for oncogene‐derived transcripts. Hum Mol Genet. 2011;20(22):4422–4429. PubMed PMC

Vicens Q, Westhof E. Biogenesis of circular RNAs. Cell. 2014;159(1):13–14. PubMed

Starke S, Jost I, Rossbach O, Schneider T, Schreiner S, Hung LH, et al. Exon circularization requires canonical splice signals. Cell Rep. 2015;10(1):103–111. PubMed

Li X, Yang L, Chen LL. The biogenesis, functions, and challenges of circular RNAs. Mol Cell. 2018;71(3):428–442. PubMed

Chen LL, Yang L. Regulation of circRNA biogenesis. RNA Biol. 2015;12(4):381–388. PubMed PMC

Zhang M, Xin Y. Circular RNAs: a new frontier for cancer diagnosis and therapy. J Hematol Oncol. 2018;11(1):21. PubMed PMC

Wu W‐L, Li S, Zhao G‐J, Li N‐Y, Wang X‐Q. Identification of circular RNAs as novel biomarkers and potentially functional competing endogenous RNA network for myelodysplastic syndrome patients. Cancer Sci. 2021;112(5):1888–1898. PubMed PMC

Merkerova MD, Klema J, Kundrat D, Szikszai K, Krejcik Z, Hrustincova A, et al. Noncoding RNAs and their response predictive value in azacitidine‐treated patients with myelodysplastic syndrome and acute myeloid leukemia with myelodysplasia‐related changes. Cancer Genomics Proteomics. 2022;19(2):205–228. PubMed PMC

Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391–2405. PubMed

Greenberg PL, Tuechler H, Schanz J, Sanz G, Garcia‐Manero G, Solé F, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood. 2012;120(12):2454–2465. PubMed PMC

Hrustincova A, Krejcik Z, Kundrat D, Szikszai K, Belickova M, Pecherkova P, et al. Circulating small noncoding RNAs have specific expression patterns in plasma and extracellular vesicles in myelodysplastic syndromes and are predictive of patient outcome. Cell. 2020;9(4):794. PubMed PMC

Kaisrlikova M, Vesela J, Kundrat D, Votavova H, Dostalova Merkerova M, Krejcik Z, et al. RUNX1 mutations contribute to the progression of MDS due to disruption of antitumor cellular defense: a study on patients with lower‐risk MDS. Leukemia. 2022;36:1898–1906. 10.1038/s41375-022-01584-3 PubMed DOI PMC

Andrews S. FastQC – a quality control tool for high throughput sequence data. Babraham Bioinformatics; 2010. Available from: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. 10.1093/bioinformatics/btu170 PubMed DOI PMC

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA‐seq aligner. Bioinformatics. 2013;29:15–21. 10.1093/bioinformatics/bts635 PubMed DOI PMC

Kovaka S, Zimin AV, Pertea GM, Razaghi R, Salzberg SL, Pertea M. Transcriptome assembly from long‐read RNA‐seq alignments with StringTie2. Genome Biol. 2019;20(1):278. PubMed PMC

Li H, Durbin R. Fast and accurate short read alignment with Burrows‐Wheeler transform. Bioinformatics. 2009;25:1754–1760. 10.1093/bioinformatics/btp324 PubMed DOI PMC

Gao Y, Zhang J, Zhao F. Circular RNA identification based on multiple seed matching. Brief Bioinform. 2018;19:803–810. 10.1093/bib/bbx014 PubMed DOI

Zhang J, Chen S, Yang J, Zhao F. Accurate quantification of circular RNAs identifies extensive circular isoform switching events. Nat Commun. 2020;11(1):90. PubMed PMC

Humphreys DT, Fossat N, Demuth M, Tam PPL, Ho JWK. Ularcirc: visualization and enhanced analysis of circular RNAs via back and canonical forward splicing. Nucleic Acids Res. 2019;47(20):e123. PubMed PMC

Szikszai K, Krejcik Z, Klema J, Loudova N, Hrustincova A, Belickova M, et al. LncRNA profiling reveals that the deregulation of H19, WT1‐AS, TCL6, and LEF1‐AS1 is associated with higher‐risk myelodysplastic syndrome. Cancers (Basel). 2020;12(10):1–21. PubMed PMC

Huang HY, Lin YCD, Li J, Huang KY, Shrestha S, Hong HC, et al. MiRTarBase 2020: updates to the experimentally validated microRNA‐target interaction database. Nucleic Acids Res. 2020;48(D1):D148–D154. PubMed PMC

Sherman BT, Hao M, Qiu J, Jiao X, Baseler MW, Lane HC, et al. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res. 2022;50(W1):W216–W221. PubMed PMC

Soen B, Vandamme N, Berx G, Schwaller J, Van Vlierberghe P, Goossens S. ZEB proteins in leukemia: friends, foes, or friendly foes? Hemasphere. 2018;2(3):e43. PubMed PMC

Kotake Y, Sagane K, Owa T, Mimori‐Kiyosue Y, Shimizu H, Uesugi M, et al. Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat Chem Biol. 2007;3(9):570–575. PubMed

Alsafadi S, Houy A, Battistella A, Popova T, Wassef M, Henry E, et al. Cancer‐associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat Commun. 2016;7(1):1–12. PubMed PMC

Liberante FG, Lappin K, Barros EM, Vohhodina J, Grebien F, Savage KI, et al. Altered splicing and cytoplasmic levels of tRNA synthetases in SF3B1‐mutant myelodysplastic syndromes as a therapeutic vulnerability. Sci Rep. 2019;9(1):2678. PubMed PMC

Hsu J, Reilly A, Hayes BJ, Clough CA, Konnick EQ, Torok‐Storb B, et al. Reprogramming identifies functionally distinct stages of clonal evolution in myelodysplastic syndromes. Blood. 2019;134(2):186–198. PubMed PMC

Brian Dalton W, Helmenstine E, Walsh N, Gondek LP, Kelkar DS, Read A, et al. Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation. J Clin Invest. 2019;129(11):4708–4723. PubMed PMC

Dudekula DB, Panda AC, Grammatikakis I, De S, Abdelmohsen K, Gorospe M. Circinteractome: a web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA Biol. 2016;13(1):34–42. PubMed PMC

Ryšavý P, Kléma J, Merkerová MD. circGPA: circRNA functional annotation based on probability‐generating functions. BMC Bioinformatics. 2022;23(1):392. PubMed PMC

Liang D, Tatomer DC, Luo Z, Wu H, Yang L, Chen LL, et al. The output of protein‐coding genes shifts to circular RNAs when the pre‐mRNA processing machinery is limiting. Mol Cell. 2017;68(5):940–954.e3. PubMed PMC

Lux S, Blätte TJ, Cocciardi S, Schwarz K, Döhner H, Döhner K, et al. Deregulated expression of circular RNAs in acute myeloid leukemia. Blood. 2018;132(Suppl 1):3894. PubMed PMC

Zhang Y, Xu L, Li A, Han X. The roles of ZEB1 in tumorigenic progression and epigenetic modifications. Biomed Pharmacother. 2019;110:400–408. PubMed

Almotiri A, Alzahrani H, Menendez‐Gonzalez JB, Abdelfattah A, Alotaibi B, Saleh L, et al. Zeb1 modulates hematopoietic stem cell fates required for suppressing acute myeloid leukemia. J Clin Invest. 2021;131(1):e129115. PubMed PMC

Xu Y, Dong X, Ma B, Mu P, Kong X, Li D. Circ_0000228 promotes cervical cancer progression via regulating miR‐337‐3p/TGFBR1 Axis. Cell J. 2022;24(2):91–98. PubMed PMC

Liu S, Li B, Li Y, Song H. Circular RNA circ_0000228 promotes the malignancy of cervical cancer via microRNA‐195‐5p/ lysyl oxidase‐like protein 2 axis. Bioengineered. 2021;12(1):4397–4406. PubMed PMC

Pei X, Zhang Y, Wang X, Xue B, Sun M, Li H. Circular RNA circ‐ZEB1 acts as an oncogene in triple negative breast cancer via sponging miR‐448. Int J Biochem Cell Biol. 2020;126:105798. PubMed

Gong Y, Mao J, Wu D, Wang X, Li L, Zhu L, et al. Circ‐ZEB1.33 promotes the proliferation of human HCC by sponging miR‐200a‐3p and upregulating CDK6. Cancer Cell Int. 2018;18(1):116. PubMed PMC

Liu W, Zheng L, Zhang R, Hou P, Wang J, Wu L, et al. Circ‐ZEB1 promotes PIK3CA expression by silencing miR‐199a‐3p and affects the proliferation and apoptosis of hepatocellular carcinoma. Mol Cancer. 2022;21(1):72. PubMed PMC

Jiang H, Yang L, Guo L, Cui N, Zhang G, Liu C, et al. Impaired mitophagy of nucleated erythroid cells leads to anemia in patients with myelodysplastic syndromes. Oxid Med Cell Longev. 2018;2018:6328051. PubMed PMC

Zhang Y, Wang M, Zang X, Mao Z, Chen Y, Mao F, et al. CircHN1 affects cell proliferation and migration in gastric cancer. J Clin Lab Anal. 2020;34(10):e23433. PubMed PMC

Catanzaro G, Besharat ZM, Carai A, Jäger N, Splendiani E, Colin C, et al. MiR‐1248: a new prognostic biomarker able to identify supratentorial hemispheric pediatric low‐grade gliomas patients associated with progression. Biomark Res. 2022;10(1):44. PubMed PMC

Du F, Guo T, Cao C. Restoration of upk1a‐as1 expression suppresses cell proliferation, migration, and invasion in esophageal squamous cell carcinoma cells partially by sponging microrna‐1248. Cancer Manag Res. 2020;12:2653–2662. PubMed PMC

Wang C, Wang B, Liang W, Zhou C, Lin W, Meng Z, et al. Hsa‐miR‐1248 suppressed the proliferation, invasion and migration of colorectal cancer cells via inhibiting PSMD10. BMC Cancer. 2022;22(1):922. PubMed PMC

Yang T, Li M, Li H, Shi P, Liu J, Chen M. Downregulation of circEPSTI1 represses the proliferation and invasion of non‐small cell lung cancer by inhibiting TRIM24 via miR‐1248 upregulation. Biochem Biophys Res Commun. 2020;530(1):348–354. PubMed