P595: DISCOVERY OF NOVEL CIRCULAR RNAS (CIRCRNAS) OF THE APOPTOSIS-RELATED BAX AND BCL2L12 GENES WITH A MULTIFACETED ROLE IN CHRONIC LYMPHOCYTIC LEUKEMIA, USING CUTTING-EDGE NANOPORE SEQUENCING TECHNOLOGY

Abstract

Background: Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key indirect regulators of gene expression, with deregulated expression and established involvement in several human malignancies. Many circRNAs have been shown to bind microRNAs (miRNAs), thus making miRNAs unavailable to regulate protein-coding gene expression. Moreover, some of them have been described to interact with chromatin and affecting its structure. Despite their pivotal roles in the development and progression of cancer, only three studies have investigated their expression and role(s) in chronic lymphocytic leukemia (CLL), so far. The products of BCL2 and its homologues, including BAX and BCL2-like 12 (BCL2L12), are implicated in CLL, as apoptosis regulators. Moreover, expression levels of both BAX and BCL2L12 mRNAs are deregulated in CLL and are associated with patients’ response to therapy and overall survival. However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their regulatory potential. Aims: We sought to further elucidate the contribution of both BAX and BCL2L12 in CLL by unraveling the identity of their circRNAs in a B-cell leukemic cell line (EHEB) and by studying in silico their potential interactions with miRNAs having an already established role in CLL. Methods: EHEB cells were propagated according to ATCC instructions. After total RNA extraction, 2μg of total RNA were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of BAX and BCL2L12 genes; thus, only cDNAs from BAX and BCL2L12 circRNAs were amplified, respectively. After having mixed and purified all nested-PCR products, nanopore sequencing libraries were built according to an optimized protocol. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA sequences were identified using an in-house–built, PERL-based algorithm. A single-molecule resolution fluorescent in situ hybridization (FISH) method called circFISH was used to visualize the circRNA distribution in the cells. Lastly, miRDB was used to predict interactions of the most abundant BAX and BCL2L12 circRNAs with miRNAs. Results: We discovered 18 and 21 novel circRNAs produced by BAX and BCL2L12 genes, respectively, in this leukemic cell line. The exon structure of these novel circRNAs showed remarkable diversity, also comprising genomic regions that have previously been considered as intronic, as they are not included in messenger RNAs (mRNAs). Interestingly, one of the two most abundant BAX circRNAs is composed of a single exon, also spanning two introns. Similar intron retentions have been observed in a few BCL2L12 circRNAs. The gamut of miRNAs predicted to be bound by novel circRNAs includes miR-484, miR-181a-2-3p and miR-214-3p. Particularly, the inhibition of miR-214-3p is associated with elevated apoptosis in leukemic B-cells. Summary/Conclusion: Alternative splicing and back-splicing mechanisms of the primary BAX and BCL2L12 transcripts produce multiple distinct circRNAs in this B-cell CLL cell line. Interestingly, one of the two most abundant circular transcripts of BAX is a circRNA spanning three exons and two intervening introns of this gene. Lastly, several miRNAs are predicted to be sequestered by circRNAs of either of these apoptosis-related genes, including miR-181 family members that have been associated with chemosensitivity. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.