In Vitro Validation of Micro-RNAs (miRNAs) Associated to Treatment Failure in Diffuse Large B-Cell Lymphoma (DLBCL)
Authors: Sas Adriana , Bento Leyre , Muncunill Josep , Martínez-Serra Jordi , Ros Teresa , Asensio Víctor , Vögler Oliver , Salar Antonio , Sampol Antonia , Alemany Regina , Gutierrez Antonio 
Published: November 2020

Introduction:
Micro-RNAs (miRNAs), have been shown to be one of the main regulators of gene expression and other biological processes. Recently, attention is focused on their potential role as prognostic predictors in different types of cancer, including diffuse large B cell lymphoma (DLBCL). We previously reported several miRNAs associated to progression/refractoriness in DLBCL (miR-1244, miR-193b-5p and miR-1231). The aim of this study was to validate these resultsin vitro, exploring the molecular pathways involved.
Material and methods:
The DLBCL cell line, U-2932 (activated B-cell (ABC) subtype) (ACC 633) was obtained from DSMZ (Germany). Transient reverse transfection of DLBCL cells for 48 or 72 h with mirVana miRNA mimics or inhibitors (miR-1244, miR-193b-5p and miR-1231) or with a miRNA negative control (100 nM) was performed using Viromer®GREEN reagent (Lipocalyx). In some experiments, after 24 h of transfection cells were treated with vehicle (1% DMSO) or with increasing concentrations of CHOP (cyclophosphamide, adriamycin, vincristine sulfate, and prednisone) (Sigma-Aldrich) for 48 h. The ratio of the four drugs was 80 mg/ 5.33 mg/ 0.16 mg/ 5.77 mg. After 48 h of transfection or treatment, cell viability was measured using the CellTiter 96®AQueous One Solution and total RNA was isolated using the RNeasy Mini Kit from Qiagen. Gene expression profiling (GEP) was performed using GeneChip human Clarion S of Affymetrix-ThermoFisher.
Results:
Reverse transfection of U-2932 with the miRNA mimic negative control (100 nM) for 48 h did not alter the cell viability compared to non-transfected (NT) cells, showing that the transfection method did not affect viability in these DLBCL cells. Transfection of mimic miR-1244, miR-193b-5p or miR-1231 (100 nM) did not significantly alter the viability compared to cells transfected with the miRNA mimic negative control. However, inhibition of these endogenous miRNA molecules for 48 h significantly decreased by 24.1 ± 5.7%, 28.9 ± 6.5% and 30.9 ± 3.3% cell viability, respectively, suggesting that these miRNAs could be involved in the survival of these tumor cells. In these experiments, the percentage of cell transfection was 89.6%. The effect of miRNA 1244, 193b-5p or 1231 on the response of DLBCL cells to CHOP treatment was also evaluated. Transfection of U-2932 cells with miRNA mimic negative control did not alter its sensibility to CHOP, which dose-dependently decreased its viability with an IC50 of 1.45 μg/ml in NT or transfected cells. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability in the presence of vehicle (1% DMSO) and also reverted the inhibitory effect of CHOP (0.3 μg/ml) after 48 h of treatment. However, transfection of U-2932 cells with mimic miR-1231 (100 nM) did not alter the inhibitory effect of CHOP (0.3, 1 and 3 μg/ml) on cell viability at any of the concentrations studied. GEP studies showed that these miRNAs reduced the expression of genes associated to chemosensitivity (MCTP1) in the case of miR-1244 and miR-193-5p or tumoral suppressor genes (NRN1) in the case of miR-1231.
Conclusions:
We validatedin vitroour previously reported miRNAs about the role of miR-1244, miR-193b-5p and miR-1231 associated to treatment failure. The inhibition of endogenous miR-1244, miR-193b-5p or miR-1231 molecules significantly decreased cell viability. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability of cells treated with CHOP. GEP studies showed that these miRNAs were linked to chemoresistance and inhibition of tumoral suppressor genes. Future works will have to translate these results to clinical practice in DLBCL.

Disclosures
Salar: Celgene:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau.