Novel circular RNAs of the apoptosis‐related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing

• Published in: FEBS open bio 2023-10, Vol.13 (10), p.1953-1966
• Main Authors: Kontos, Christos K., Karousi, Paraskevi, Artemaki, Pinelopi I., Abdelgawad, Ahmed, Dimitriadou, Aspasia, Machairas, Nikolaos P., Sideris, Diamantis C., Pappa, Vasiliki, Scorilas, Andreas, Batish, Mona, Papageorgiou, Sotirios G.
• Format: Article
• Language: English
• Subjects: Apoptosis; BAX protein; Bcl-2 protein; BCL2 family; Biomarkers; Blood & organ donations; Blood donors; Chronic lymphocytic leukemia; circRNA; Circular RNA; DNA polymerase; Fluorescence in situ hybridization; Gene expression; Genes; Hematological Cancer; Hexamers; Investigations; Leukemia; Leukocytes (mononuclear); Localization; Lymphocytes B; MicroRNAs; Non-Coding RNA; Pathogenesis; Peripheral blood mononuclear cells; Polymerase chain reaction; pro‐apoptotic; Ribonucleic acid; RNA; RNA Sequencing; single‐molecule fluorescence in situ hybridization; third generation (long‐read) sequencing; transcriptomics
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1002/2211-5463.13672

Circular RNAs (circRNAs), a novel RNA type generated by back‐splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non‐leukemic blood donors was extracted and reverse‐transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first‐strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non‐leukemic blood donors. Lastly, a single‐molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non‐leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B‐cell CLL. Novel circular RNAs (circRNAs) deriving from BAX and BCL2L12 were discovered using nanopore sequencing. These circRNAs exhibit diverse exon structures, distinct intracellular localization, and may interact with microRNAs with a proven role in chronic lymphocytic leukemia (CLL). A complex circRNA expression pattern in CLL patients and non‐leukemic blood donors was found as well. Our findings suggest a multifaceted role of these circRNAs in B‐cell CLL.