Tumor-intrinsic miR-21a orchestrates Treg-mediated immunosuppression through Pag-1 repression

Immune checkpoint blockades (ICBs) such as anti-PD-1 and anti-CTLA-4 achieve durable responses in some cancers but fail in many patients, highlighting the need for additional immune modulators. Through comparative small RNA profiling of ICB-treated mouse tumors, we identified miR-21a as highly upregulated during combination immunotherapy. To define its tumor-intrinsic function, we generated CRISPR-Cas9 miR-21a knockout (KO) models in melanoma (B16F10, YUMM1.7), colorectal (MC38, CT26), and triple-negative breast cancer (4T1). Despite unchanged in vitro proliferation, miR-21a KO tumors exhibited markedly reduced growth in vivo, even without anti-PD-1 therapy, demonstrating its broad pro-tumorigenic role across cancer types. Single-cell RNA sequencing of MC38 KO tumors revealed reprogramming of regulatory T cells (Tregs) toward a less suppressive phenotype. Mechanistically, Pag-1 (phosphoprotein associated with glycosphingolipid microdomains 1) was identified as a direct target of miR-21a, and its de-repression in KO tumors contributed to enhanced immune activation. These findings uncover miR-21a as a conserved tumor-intrinsic immune regulator that orchestrates Treg-mediated immunosuppression through Pag-1, providing a promising therapeutic axis to improve immunotherapy efficacy.