17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Abstract

Introduction: We previously performed global miRNA expression profiling of cHL vs. non-Hodgkin lymphoma (NHL) cell lines and sorted germinal centre B-cells (GCB). In the group of 79 deregulated miRNAs (p < 0.05) we became particularly interested in the upregulated miR-23a-3p, a potential oncomiR, predicted to regulate the expression of TNFAIP3 and the downregulated miR-148a-3p, a potential tumor suppressor miRNA, predicted to regulate the expression of IL15. Therefore, we aimed to decipher the role of miR-23a-3p and miR-148a-3p in cHL pathogenesis. Methods: Expression analyses were performed with TaqMan Advanced miRNA Assays versus two miRNA controls. Laser capture microdissection was used for primary HRS collection. 1,000 (miRNA expression) and 2 × 200 (DNA methylation) HRS cells were collected per case. Bisulfite pyrosequencing assay was designed using the PyroMark Software and sequencing performed using the PyroMark Q24 sequencer. MiRNA-mRNAs interactions were validated with Dual-Glo Luciferase Assay using respective miRNA mimics. For overexpression pre-miRNAs were cloned into the pCDH-CMV-MCS-EF1α-GreenPuro vector and transductions were performed in triplicate. Cell proliferation was measured using CCK-8 in four replications in three independent reactions by the GloMax®96 Microplate reader. Results: We confirmed the overexpression of miR-23a-3p (p < 0.001) and downregulation of miR-148a-3p (p < 0.014) in microdissected HRS cells (n = 10) vs. GCB cells (n = 10). Moreover, we observed inverse correlation of miR-148a-3p expression with DNA methylation level of an adjacent CpG island (r = −0.72, p < 0.01; cHL n = 7, NHL n = 10). Elevated DNA methylation of this CpG island was detected also in 2/6 cHL primary cases. Next, we validated interactions between miR-23a-3p and miR-148a-3p and their target genes. A reduction by 34% (p < 0.001) and 40% (p < 0.01) of the luciferase signal was observed in consequence of the interaction of miR-23a-3p mimic—TNFAIP3 3’UTR and miR-148a-3p mimic—IL15 3’UTR, respectively. Moreover, the abundance of TNFAIP3 protein was reduced after overexpression of the miR-23a (coexpressed with miR-27a) in L428 (p = 0.0018) and GCB6-16 (p = 0.0083) cell lines, compared to empty vector transductions. Whereas overexpression of miR-148a in three cHL cell lines caused a 32% decrease (p < 0.05) in proliferation in the KM-H2 cell line compared to empty vector transductions. The research was funded by: This research was funded by the National Science Center, Poland grants: 2014/14/M/NZ2/00529 to M.G. and the 2019/32/T/NZ5/00441 Etiuda scholarship to JP. SH is supported by the German Research Foundation (DFG, HA6145/3-1). MG, ADz-K and NR received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952304. Keywords: Hodgkin lymphoma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.