TNFα protein, DNA methylation, mRNA and miRNA expression evaluation in multiple sclerosis

Background Tumor necrosis factor alpha (TNF alpha) is a key cytokine involved in the inflammatory and neurodegenerative processes underlying multiple sclerosis (MS). Its expression is finely regulated by epigenetic and post-transcriptional mechanisms, including promoter methylation and miRNA activity. The objective of this study is to investigate TNF alpha expression, promoter methylation, and its regulation by miR-130a-3p in patients with relapsing-remitting (RR)MS, evaluating both serum and saliva as potential diagnostic biofluids.Methods RRMS patients in clinical remission and sex and age-matched healthy controls (HC) were enrolled. TNF alpha levels were quantified in serum, peripheral blood mononuclear cell (PBMC) supernatants, and saliva using ELISA. TNF alpha mRNA expression and promoter methylation were analyzed by qPCR and pyrosequencing, respectively. Bioinformatic tools (TargetScan, miRTargetLink 2.0, miEAA 2) were used to explore miRNA-TNF alpha interactions, and miR-130a-3p expression was evaluated in serum and saliva by qPCR.Results RRMS patients showed significantly higher TNF alpha mRNA and protein levels compared to HC, paralleled by significant hypomethylation of the TNF alpha promoter in PBMCs. miR-130a-3p was markedly downregulated in both serum and saliva, exhibiting an inverse trend with TNF alpha expression. Salivary TNF alpha levels mirrored serum alterations, supporting the feasibility of saliva as a noninvasive biomarker source.Conclusion The data indicate that TNF alpha upregulation in RRMS is associated with promoter hypomethylation and reduced miR-130a-3p expression, suggesting a coordinated epigenetic and post-transcriptional control of this cytokine. The parallel trends observed in saliva and serum highlight the potential use of salivary TNF alpha and miR-130a-3p as minimally invasive biomarkers for MS monitoring and early diagnosis.