Rab27a+CAF exosomal miR-9-5p promotes osteosarcoma progression via CREBRF/MAPK signaling pathway

Abstract

Osteosarcoma is the most common primary malignant bone tumor in adolescents, characterized by high rates of recurrence and chemotherapy resistance. Exosomes, as key mediators of intercellular communication, have emerged as potential biomarkers and therapeutic targets in cancer. However, the mechanisms by which cancer-associated fibroblasts (CAFs) exosomal miRNAs regulate osteosarcoma progression remain unclear. This study investigates the role of exosomal miR-9-5p derived from Rab27a⁺CAF in promoting osteosarcoma malignancy via CREBRF/MAPK (Mitogen-Activated Protein Kinase) signaling axis. Serum and tissue derived exosomes were isolated, followed by small RNA sequencing and validation of miR-9-5p expression using qRT-PCR (quantitative RT-PCR). Single-cell RNA sequencing was performed on osteosarcoma and adjacent tissues to characterize the tumor microenvironment. The interaction between exosomal miR-9-5p and CREBRF was confirmed using dual-luciferase reporter assays. CCK8 (Cell Counting Kit 8), Edu (5-Ethynyl-2′-deoxyuridine), cell cycle, and cell apoptosis in vitro were conducted to explore the function of exosomal miR-9-5p/CREBRF/MAPK signaling pathway on osteosarcoma. The tumorigenicity of CAF exosomal miR-9-5p was assessed using a subcutaneous osteosarcoma mouse model. Exosomal miR-9-5p was significantly upregulated in serum and tissue from osteosarcoma patients and was associated with poor prognosis. Single-cell RNA sequencing revealed that Rab27a⁺CAF were major contributors in the tumor microenvironment. Functional assays demonstrated that exosomal miR-9-5p promoted proliferation, cisplatin resistance, cell cycle progression, and inhibited apoptosis in osteosarcoma cells. Mechanistically, miR-9-5p targeted and downregulated CREBRF, thereby activating the MAPK signaling pathway. Experiments in vivo confirmed that high levels of exosomal miR-9-5p enhanced tumor growth and MAPK pathway activity. This study identifies Rab27a⁺CAF exosomal miR-9-5p as a key oncogenic factor in osteosarcoma, which promotes tumor progression and chemoresistance by suppressing CREBRF and activating the MAPK pathway. These findings highlight exosomal miR-9-5p as a promising biomarker and therapeutic target for precision treatment of osteosarcoma.