Milan Stefanović, Ivan Jovanović, Nađa Trklja*, Jovana Kuveljić, Aleksandra Stanković and Maja Živković
Vinča Institute of Nuclear Sciences, National Institute of the Republic of Serbia, University of Belgrade, Department of Radiobiology and Molecular Genetics
nadja.trklja [at] vin.bg.ac.rs
Abstract
Chronic active lesions (CAL) are the main hallmarks of progressive multiple sclerosis (progMS) and are characterized by a demyelinated, inactive core surrounded by an iron-enriched rim. Change of lesion phenotype into stable inactive lesions (IL), gliotic white mater scars, relates to iron loss. Recently, ferroptosis, a form of iron-dependent programmed cell death characterized by iron overload, has been linked to MS. Therefore, we aimed to identify genes associated with ferroptosis relevant to separate CAL from IL, by analyzing total transcriptome data from post-mortem brain lesions of progMS patients. Whole RNA data (containing mRNA and miRNA counts) from GEO database GSE138614 (16 CAL and 14 IL lesions from 10 progMS patients) were analyzed. Weighted Gene Coexpression Network Analysis (WGCNA) was performed, identifying expression modules most correlated with IL (Tan: r=0.6, p=0.0033, ngenes=301; Blue: r=-0.66, p=0.0009, ngenes=2861). Using DESeq2 (Log2FC>|0.6|, p.adj<0.05) we’ve identified 2728 differentially expressed genes (DEGs) coding both proteins and miRNAs in the IL vs. CAL comparison. We’ve intersected DEGs, ferroptotsis-related genes from human biological sources (FerrDbV3: n=1196) and the selected WGCNA module genes to extract ferroptotsis-related genes associated with IL (n=43; 17 ferroptosis drivers, 19 suppressors and 5 with dual roles). STRING (interaction score≥0.4; all interactions) was used to identify protein interactions between the 43 identified ferroptotsis-related genes. Interaction networks were analyzed via Cytoscape plug-in cytoHubba. PKM, MDH2, GOT1, PPARGC1A, PGAM1, KCNA1 and CKB were identified as the most interconnected, hub, protein-coding genes. By employing miRTarBase-v9.0, we’ve identified miRNAs coded by DEGs MIR612, MIR6852 and MIR17HG as potential regulators of key ferroptosis-related genes. MIR17HG-coded miRNAs had the greatest regulatory potential (targeting 14 key genes) while being a key ferroptosis-related gene and known suppressor of ferroptosis. Every hub gene and potential miRNA regulator had a Receiver Operating Characteristic Area Under Curve value ≥0.8, meaning that they were valid discriminators between CAL and IL. We conclude that the protein-coding genes PKM, MDH2, GOT1, PPARGC1A, PGAM1, KCNA1, and CKB, along with the miRNA host gene MIR17HG, potentially contribute to lesion phenotype change to IL in progMS through ferroptosis-related mechanisms. Further experiments are needed to validate this finding.
Keywords: Ferroptosis, multiple sclerosis, lesions, progression