miR-221-3p mediates T-cell differentiation through repressing DYRK1A

Objective This study aims to comprehensively investigate the role and underlying molecular mechanism of hsa-miR-221-3p in regulating the differentiation and effector functions of distinct T cell subpopulations, and to further explore the potential implication of modulating miR-221-3p activity for optimizing CAR-T cell therapy persistence. Methods Based on miRNA expression profiles of four T cell subpopulations [naïve T cells (TN), stem cell-like memory T cells (TSCM), central memory T cells (TCM), and effector memory T cells (TEM)] obtained by high-throughput sequencing in a previous study, differential expression analysis was performed between TN and other subpopulations to identify hsa-miR-221-3p. Computational prediction was used to screen putative targets of miR-221-3p, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A dual luciferase reporter assay was conducted to verify the direct binding of miR-221-3p to DYRK1A . Functional experiments were performed to assess the effects of miR-221-3p overexpression on T cell proliferation, apoptosis, cell death, cytokine secretion (TNF- α and IFN- γ ), and expression of differentiation-related genes. Flow cytometry was used to analyze the changes in T cell subsets (marked by CD62L and CD45RO) after miR-221-3p overexpression. Results GO and KEGG analyses revealed that miR-221-3p target genes were enriched in transcription regulatory pathways. Dual-luciferase assays confirmed direct binding of miR-221-3p to DYRK1A , suppressing its expression in T cells. Functionally, miR-221-3p overexpression reduced T cell proliferation, increased apoptosis, and enhanced TNF- α and IFN- γ secretion. Mechanistically, it downregulated early differentiation genes while upregulating late differentiation/effector genes ( Blimp-1 , T-bet , GZMB , GNLY ). Flow cytometry confirmed a shift from TN toward TEM/TEFF subsets. Conclusion miR-221-3p drives terminal effector differentiation of T cells by repressing DYRK1A . Inhibition of miR-221-3p preserves early memory subsets, offering a potential strategy to enhance CAR-T cell persistence.