Department of Bone and Soft Tissue Oncology, Shanxi Province Cancer Hospital/ Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, Shanxi, China.
Department of Radiotherapy, Shanxi Province Cancer Hospital/ Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, Shanxi, China.
Protein Pept Lett. 2024;31(7):559-568. doi: 10.2174/0109298665314658240712051206.
Osteosarcoma (OS) is the leading cancer-associated mortality in childhood and adolescence. Increasing evidence has demonstrated the key function of microRNAs (miRNAs) in OS development and chemoresistance. Among them, miRNA-605-3p acted as an important tumor suppressor and was frequently down-regulated in multiple cancers. However, the function of miR-650-3p in OS has not been reported.
The aim of this work is to explore the novel role of miR-605-3p in osteosarcoma and its possible involvement in OS chemotherapy resistance.
The expression levels of miR-605-3p in OS tissues and cells were assessed by reverse transcription quantitative PCR (RT-qPCR). The relevance of miR-605-3p with the prognosis of OS patients was determined by the Kaplan-Meier analysis. Additionally, the influence of miR-605-3p on OS cell growth was analyzed using the cell counting kit-8, colony formation assay, and flow cytometry. The mRNA and protein expression of RAF1 were detected by RT-qPCR and western blot. The binding of miR-605-3p with the 3'-UTR of RAF1 was confirmed by dual-luciferase reporter assay.
Our results showed that miR-605-3p was markedly decreased in OS tissues and cells. A lower level of miR-605-3p was strongly correlated with lymph node metastasis and poor 5-year overall survival rate of OS patients. assay found that miR-605-3p suppressed OS cell proliferation and promoted cell apoptosis. Mechanistically, the proto-oncogene RAF1 was seen as a target of miR-605-3p and strongly suppressed by miR-605-3p in OS cells. Restoration of RAF1 markedly eliminated the inhibitory effect of miR-605-3p on OS progression, suggesting RAF1 as a key mediator of miR-605-3p. Consistent with the decreased level of RAF1, miR-605-3p suppressed the activation of both MEK and ERK in OS cells, which are the targets of RAF1. Moreover, lower levels of miR-605-3p were found in chemoresistant OS patients, and downregulated miR-605-3p increased the resistance of OS cells to therapeutic agents.
Our data revealed that miR-605-3p serves as a tumor suppressor gene by regulating RAF1 and increasing the chemosensitivity of OS cells, which provided the novel working mechanism of miR-605-3p in OS. Engineering stable nanovesicles that could efficiently deliver miR-605-3p with therapeutic activity into tumors could be a promising therapeutic approach for the treatment of OS.
骨肉瘤（OS）是儿童和青少年癌症相关死亡的主要原因。越来越多的证据表明，微小 RNA（miRNA）在 OS 发展和化疗耐药性中起着关键作用。其中，miRNA-605-3p 作为一种重要的肿瘤抑制因子，在多种癌症中经常下调。然而，miR-650-3p 在 OS 中的功能尚未报道。
本研究旨在探讨 miR-605-3p 在骨肉瘤中的新作用及其在 OS 化疗耐药中的可能参与。
采用逆转录定量 PCR（RT-qPCR）检测 OS 组织和细胞中 miR-605-3p 的表达水平。通过 Kaplan-Meier 分析确定 miR-605-3p 与 OS 患者预后的相关性。此外，通过细胞计数试剂盒-8（CCK-8）、集落形成实验和流式细胞术分析 miR-605-3p 对 OS 细胞生长的影响。采用 RT-qPCR 和 Western blot 检测 RAF1 的 mRNA 和蛋白表达。通过双荧光素酶报告基因实验证实 miR-605-3p 与 RAF1 3'-UTR 的结合。
我们的研究结果表明，miR-605-3p 在 OS 组织和细胞中明显下调。较低水平的 miR-605-3p 与 OS 患者的淋巴结转移和不良 5 年总生存率强烈相关。CCK-8 实验发现，miR-605-3p 抑制 OS 细胞增殖并促进细胞凋亡。机制上，原癌基因 RAF1 被视为 miR-605-3p 的靶标，并在 OS 细胞中被 miR-605-3p 强烈抑制。RAF1 的恢复显著消除了 miR-605-3p 对 OS 进展的抑制作用，表明 RAF1 是 miR-605-3p 的关键介导因子。与 RAF1 水平降低一致，miR-605-3p 抑制了 OS 细胞中 RAF1 的靶点 MEK 和 ERK 的激活。此外，在化疗耐药的 OS 患者中发现 miR-605-3p 水平较低，下调 miR-605-3p 增加了 OS 细胞对治疗药物的耐药性。
我们的数据表明，miR-605-3p 通过调节 RAF1 并增加 OS 细胞的化疗敏感性，作为一种肿瘤抑制基因发挥作用，为 miR-605-3p 在 OS 中的作用提供了新的工作机制。工程化能够将具有治疗活性的稳定纳米囊泡有效递送至肿瘤的 miR-605-3p 可能成为治疗 OS 的一种有前途的治疗方法。
