Niu Xiaoying, Tian Wen, Li Zhongyi, Zhang Guorui, Zhang Peng
Bone and Soft tissue department, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou 450008, China.
Bone and Soft tissue department, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, Zhengzhou 450008, China; School of Public Health, Zhengzhou University, Henan 450001, China.
Cell Signal. 2025 Oct;134:111964. doi: 10.1016/j.cellsig.2025.111964. Epub 2025 Jun 26.
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 Rab27aCAF 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 Rab27aCAF 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 Rab27aCAF 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.
骨肉瘤是青少年中最常见的原发性恶性骨肿瘤，其特征是复发率高和化疗耐药。外泌体作为细胞间通讯的关键介质，已成为癌症潜在的生物标志物和治疗靶点。然而，癌症相关成纤维细胞（CAFs）外泌体miRNAs调节骨肉瘤进展的机制仍不清楚。本研究探讨Rab27aCAF来源的外泌体miR-9-5p通过CREBRF/丝裂原活化蛋白激酶（MAPK）信号轴促进骨肉瘤恶性进展的作用。分离血清和组织来源的外泌体，随后进行小RNA测序，并使用定量逆转录聚合酶链反应（qRT-PCR）验证miR-9-5p的表达。对骨肉瘤及相邻组织进行单细胞RNA测序以表征肿瘤微环境。使用双荧光素酶报告基因测定法确认外泌体miR-9-5p与CREBRF之间的相互作用。进行体外细胞计数试剂盒8（CCK8）、5-乙炔基-2'-脱氧尿苷（Edu）、细胞周期和细胞凋亡实验，以探究外泌体miR-9-5p/CREBRF/MAPK信号通路对骨肉瘤的作用。使用皮下骨肉瘤小鼠模型评估CAF外泌体miR-9-5p的致瘤性。外泌体miR-9-5p在骨肉瘤患者的血清和组织中显著上调，且与预后不良相关。单细胞RNA测序显示Rab27aCAF是肿瘤微环境的主要贡献者。功能测定表明，外泌体miR-9-5p促进骨肉瘤细胞增殖、顺铂耐药、细胞周期进程并抑制细胞凋亡。机制上，miR-9-5p靶向并下调CREBRF，从而激活MAPK信号通路。体内实验证实，高水平的外泌体miR-9-5p增强肿瘤生长和MAPK通路活性。本研究确定Rab27aCAF外泌体miR-9-5p是骨肉瘤中的关键致癌因子，其通过抑制CREBRF和激活MAPK通路促进肿瘤进展和化疗耐药。这些发现突出了外泌体miR-9-5p作为骨肉瘤精准治疗的有前景的生物标志物和治疗靶点。
