A predictive serum miRNA signature impacts diffuse large B-cell lymphoma cell viability via inhibition of EGLN1 and TXNRD1 regulators of ferroptosis

Abstract

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disorder. Prognostic factors include genomic alterations and cell-of-origin (COO) subtypes, even though they cannot fully predict treatment response. MicroRNAs (miRNAs) deregulated in patient tumours and blood are promising non-invasive biomarkers. Several circulating miRNAs were found to be correlated with progression-free survival (PFS), independently of other prognosticators. However, miRNA signatures, rather than individual miRNAs, represent more reliable biomarkers and a better mirror of the disease. In this study, we identified circulating miRNAs differentially expressed between R-CHOP refractory and responding subjects by small-RNA sequencing on serum from 33 DLBCL patients. Among the identified miRNAs, the combined expression of three of them improved the predictive performance and was correlated with PFS. Two out of three miRNAs, miR-421 and miR-324-5p, were also differentially expressed in tumour tissues based on treatment response. Overexpressing these miRNAs reduced cell proliferation, viability and resistance to R-CHOP in the germinal centre B-like COO subtype. EGLN1 and TXNRD1, regulators of oxygen metabolism and redox homeostasis, were identified as miRNA targets and the silencing or inhibition of these genes impaired cell viability and induced ferroptosis. These results support the application of a two-miRNA signature and its targets for novel combined therapeutic interventions in DLBCL.

Keywords: circulating microRNAs; diffuse large B‐cell lymphoma; ferroptosis regulators; predictive biomarkers.

FIGURE 1

Genome‐wide analysis and selection of a serum miRNA‐signature deregulated in diffuse large B‐cell lymphoma (DLBCL) patients based on treatment response. (A) Experimental workflow for the miRNA profiling; boxplots of the indicated serum miRNA levels in non‐responsive (NR) versus responsive (R) patients. (B) Fold change expression (FC) and area under the ROC curve (AUC) of the selected five miRNAs. ROC curves for predictive accuracy of the combinations of the downregulated and upregulated miRNAs. Kaplan–Meier plots for PFS based on the miR‐200c/miR‐324/miR‐421 combination (log‐rank test). (C) Plots of the indicated miRNA levels in tumour tissues of NR versus R patients. *p ≤ 0.05; ***p ≤ 0.001 (Mann–Whitney test).

FIGURE 2

Impact of miR‐421 and miR‐324 ectopic expression on diffuse large B‐cell lymphoma (DLBCL) proliferation and viability. (A) Cell proliferation of the indicated DLBCL cells after transfection with individual or combined miRNA‐mimics. Values are reported as fold change of the viable cells over the control. (B) Cell viability after miRNA‐mimic or control‐mimic transfection. The percentage of dead cells is expressed as fold change over the control. (C) miRNA levels in the extracellular fraction (EC) and dose–response curves of the indicated DLBCL cells after treatment with CHOP plus rituximab. EC values, normalized by the intracellular miRNA expression (IC) and by cell density, are reported as the mean of at least three experiments; bars represent the standard deviation. (D) Cytotoxicity assay of the indicated DLBCL cells after transfection with individual or combined miRNA‐mimics, and upon treatment with CHOP plus rituximab, or with the drug vehicle (DMSO). Cell death is expressed as fluorescent green area normalized by the total area of confluence; representative images of cell samples after 72h of treatment are reported; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 (Student's t‐test).

FIGURE 3

Identification and validation of EGLN1 and TXNRD1 as miR‐421 and miR‐324 direct targets. (A) Venn diagrams of putative targets selected by TargetScan (blue) and miRDB (green). (B) Scheme of the biological processes impacted by EGLN1 and TXNRD1. (C) EGLN1 and TXNRD1 mRNA and protein levels in diffuse large B‐cell lymphoma (DLBCL) cells after miRNA overexpression. Values are reported as fold change over the control. (D) Scheme of putative binding sites for miRNAs in the 3′UTR of their target genes and Firefly luciferase activity. Results are normalized by basal activity of luciferase empty vector and expressed as fold activation over the control. Bars indicate the standard deviation; *p ≤ 0.05, **p ≤ 0.01 (Student's t‐test).

FIGURE 4

Impact of miR‐421/miR‐324 signature and its targets on ferroptosis. (A) mRNA and protein levels with densitometric analysis of GPX4 upon overexpression of miR‐421 and miR‐324 or the negative control in diffuse large B‐cell lymphoma (DLBCL) cells. (B) EGLN1 and TXNRD1 protein levels after gene silencing in GCB‐DLBCL cells; cell viability after EGLN1 and TXNRD1 siRNA or negative control transfection. The percentage of dead cells is expressed as fold change over the control. (C) Protein levels and densitometric analysis of the GPX4 ferroptosis marker after EGLN1 and TXNRD1 silencing in the GCB‐DLBCL cells. (D) GPX4 mRNA and protein levels with densitometric analysis in GCB‐DLBCL cells treated with 10 μM molidustat or 0.4 μM auranofin versus the DMSO treated negative control. Values are reported as fold change over the control; bars represent the standard deviation. (E) Cell viability of DLBCL cells after transfection with miRNA‐mimics and upon treatment with 2 μM Ferrostatin‐1 (Fer‐1), or with the drug vehicle (DMSO); *p ≤ 0.05 (Student's t‐test).