Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines

PLOS ONE RESEARCH ARTICLE Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines Adam Ustaszewski ID1, Julia Paczkowska1, Joanna Janiszewska1, Stephan H. Bernhart2, Julia Bein3, Núria Russiñol4, Martin-Leo Hansmann5,6, Vicente Chapaprieta ID4, José I. Martı́n-Subero4,7,8,9, Reiner Siebert10, Sylvia Hartmann3, Maciej Giefing ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Institute of Human Genetics, Polish Academy of Sciences Poznan, Poznan, Poland, 2 Interdisciplinary Center for Bioinformatics, Transcriptome Bioinformatics, University of Leipzig, Leipzig, Germany, 3 Dr. Senckenberg Institute of Pathology, Goethe University Frankfurt, Frankfurt am Main, Germany, 4 Institut d’Investigacions Biomèdiques August Pi I Sunyer, IDIBAPS, Barcelona, Spain, 5 Frankfurt Institute of Advanced Studies, Frankfurt am Main, Germany, 6 Institute of General Pharmacology and Toxicology, Goethe University Frankfurt, Frankfurt am Main, Germany, 7 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Pamplona, Spain, 8 Facultat de Medicina, Hospital Clı́nic de Barcelona and Departament de Fonaments Clı́nics, Universitat de Barcelona, Barcelona, Spain, 9 Institució Catalana de Recerca i Estudis Avançats, ICREA, Barcelona, Spain, 10 Institute of Human Genetics, Ulm University and Ulm University Medical Center, Ulm, Germany * OPEN ACCESS Citation: Ustaszewski A, Paczkowska J, Janiszewska J, Bernhart SH, Bein J, Russiñol N, et al. (2023) Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines. PLoS ONE 18(3): e0283186. https://doi.org/ 10.1371/journal.pone.0283186 Editor: Benigno C. Valdez, The University of Texas MD Anderson Cancer Center, UNITED STATES Received: May 12, 2022 Accepted: March 3, 2023 Published: March 24, 2023 Copyright: © 2023 Ustaszewski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The research was funded in whole or in part by the National Science Center, Poland (grant 2020/39/B/NZ2/01004 to MG); the European Union’s Horizon 2020 research and innovation program under grant agreement no 952304 (acronym NEXT_LEVEL) within Twinning Action; the Deutsche Forschungsgemeinschaft (grant number HA6145/3-1 to SH). For the purpose of Abstract MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788–212678849 and chr5:168090507–168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1. PLOS ONE | https://doi.org/10.1371/journal.pone.0283186 March 24, 2023 1 / 17 PLOS ONE Open Access, the author has applied a CC-BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. Competing interests: The authors declare no competing interests. Novel miRNAs in classic Hodgkin lymphoma Introduction MicroRNAs (miRNAs) play a key role in posttranscriptional regulation of gene expression, and are essential regulators of various cellular processes [1,2]. These short RNAs incorporated into the RISC complex bind to the complementary 3’UTR fragment of a target mRNA leading to its degradation or inhibition of translation [3]. Aberrant miRNA expression in classic Hodgkin lymphoma (cHL), a B-cell lymphoma characterized by the presence of few large neoplastic Hodgkin and Reed-Sternberg (HRS) cells, was first confirmed in the early 2000s [4]. By now, the role of several oncomiRs and tumor suppressor miRNAs was described in cHL [5–7]. These include the deregulated miR-155 and miR-196a and miR-23a-3p, which are assumed to contribute to the constitutive NFκB hyperactivation, a process crucial in cHL pathogenesis [6,8,9]. Moreover, also other miRNAs deregulated in cHL such as miR-9, miR-138, miR-150 were described as candidates involved in processes responsible for Hodgkin lymphomagenesis such as immune evasion and impaired B-cell receptor signalling [9]. In our recent study we have performed high throughput screening of miRNA expression in 7 widely used cHL cell lines and ten non-Hodgkin lymphoma cell lines (NHL) by RNA-seq [9]. Here we mined this dataset with the aim to find miRNAs expressed in cHL and not yet reported in miRBase (release 22.1). This brought us to the identification of two molecules with typical miRNA characteristics which are recurrently expressed in cHL cell lines. Moreover, our results suggest that the aberrant expression of these novel miRNAs is at least to some extend caused by the loss of epigenetic control in cHL cell lines. Materials and methods CHL cell lines, NHL cell lines and primary cHL samples 7 cHL cell lines (L-428 [10], HDLM-2 [11], KM-H2 [12], L-1236 [13], SUP-HD1 [14], U-HO1 [15], L-540 [16]) and 10 NHL cell lines (Burkitt lymphoma: Raji [17], Ca46 [18], Daudi [19], Namalwa [20], Ramos [21]; Diffuse Large Cell Lymphoma: OCI-LY1 [22], OCI-LY3 [22], OCI-LY7 [22], SU-DHL-6 [23]; B Cell Lymphoma: Val [24]) were used. Three cHL cell lines (HDLM-2, SUP-HD1, U-HO1) and ten NHL cell lines (Burkitt lymphoma: Raji, Ca46, Daudi, Namalwa, Ramos; Diffuse Large Cell Lymphoma: OCI-LY1, OCI-LY3, OCI-LY7, SU-DHL-6; B Cell Ly (...truncated)