miR-142-5p promotes TSCM differentiation and suppresses progressive T-cell maturation via targeting PRKCB

Adoptive cellular immunotherapy (ACT) such as CAR‑T therapy holds promise for cancer treatment. However, genetically engineered T cells often undergo terminal differentiation during ex vivo expansion, which limits their persistence and antitumor efficacy in vivo. Early‑differentiated T‑cell subsets exhibit better survival and proliferative capacity after infusion. In our previous work, we isolated four T‑cell subsets at different differentiation stages: naïve T cells (T_N), stem cell‑like memory T cells (TSCM), central memory T cells (T_CM), and effector memory T cells (T_EM), and obtained their miRNA expression profiles via high‑throughput sequencing. In the present study, we found that hsa‑miR‑142‑5p is highly expressed in TSCM cells and gradually decreases during T‑cell differentiation.

Bioinformatics analysis suggested that miR‑142‑5p target genes are involved in transcription regulation. We identified four candidate targets through reverse enrichment analysis. Dual‑luciferase reporter assays were used to validate direct targets. Functional studies were then performed in T cells overexpressing miR‑142‑5p to assess expression of differentiation‑associated and effector‑related genes, surface markers (CCR7, CD62L, CD95), cell subset proportions (T_N, T_SCM, T_EM/T_EFF), proliferation, apoptosis, and cytokine secretion (TNF‑α, IFN‑γ).

Dual‑luciferase reporter assays confirmed PRKCB as a direct target of miR‑142‑5p, and miR‑142‑5p suppressed PRKCB expression in T cells. miR‑142‑5p overexpression upregulated early differentiation‑associated genes (LEF1, CD62L, CCR7) and the anti‑apoptotic gene BCL2, while downregulating late differentiation‑associated genes (KLRG1, EOMES, PDCD1) and effector function‑related genes (GZMB, PRF1). Consistently, it enhanced early differentiation markers (CCR7, CD62L) and reduced the late marker CD95. It also increased T_N and T_SCM proportions while decreasing T_EM/T_EFF cells. Additionally, miR‑142‑5p promoted T‑cell proliferation, reduced apoptosis, and suppressed TNF‑α and IFN‑γ secretion.

In summary, miR‑142‑5p inhibits progressive T‑cell differentiation by directly targeting PRKCB, helping maintain an early‑differentiated phenotype. This offers a potential strategy to improve the persistence and efficacy of adoptive T‑cell‑based immunotherapies.