Tumor‐Derived Exosomal <scp>piR</scp> ‐hsa‐28212 Promotes Lymphatic Metastasis in Breast Cancer

Lymph node (LN) metastasis is a critical indicator of poor prognosis in breast cancer (BC). BC-derived exosomes influence intercellular communication within the tumor microenvironment, driving metastatic progression. However, the precise mechanisms by which exosomes facilitate LN metastasis in BC remain unclear. We performed small RNA sequencing on serum exosomes to identify Piwi-interacting RNAs (piRNAs) associated with BC LN metastasis. Functional investigations of exosomal piR-hsa-28212 included in vitro assays for migration and tube formation of human lymphatic endothelial cells (HLECs), alongside an in vivo footpad-popliteal LN metastasis model. Specific interactions between piR-hsa-28212 and TBX1 (T-box transcription factor 1), as well as TBX1 and VEGF receptor 3 (VEGFR3), were validated through luciferase reporter assays. The stability of TBX1 mRNA was analyzed by actinomycin D assay. RNA pulldown, RNA immunoprecipitation (RIP), and RNA fluorescence in situ hybridization (FISH) assays were conducted to explore the interaction between piR-hsa-28212 and METTL3. PiR-hsa-28212 was significantly upregulated in serum exosomes of BC patients with LN metastasis. Exosomal piR-hsa-28212 enhanced HLECs migration and tube formation in vitro and promoted lymphangiogenesis and LN metastasis in vivo. Mechanistically, exosomal piR-hsa-28212 transferred from BC cells to HLECs stabilized TBX1 mRNA, thereby upregulating VEGFR3 expression. In BC cells, piR-hsa-28212 directly bound to and stabilized METTL3 protein, modulating N6-methyladenosine (m6A) methylation of vascular endothelial growth factor C (VEGFC) mRNA and consequently increasing VEGFC expression and secretion. Collectively, these findings reveal an exosome-mediated piRNA regulatory mechanism that synergistically amplifies VEGFC/VEGFR3 signaling to drive LN metastasis in BC, highlighting piR-hsa-28212 as a potential therapeutic target. Trial Registration: KYLL-2022-338.