Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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. In vitro 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.
CONCLUSIONS: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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. In vitro 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.
CONCLUSIONS: 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.
摘要：
背景：骨肉瘤（OS）是儿童和青少年时期主要的癌症相关死亡率。越来越多的证据已经证明了microRNAs（miRNAs）在OS发育和化学抗性中的关键功能。其中,miRNA-605-3p是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中的功能尚未见报道。
目的：这项工作的目的是探讨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。
方法：通过逆转录定量PCR（RT-qPCR）评估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,miR-605-3p对OS细胞生长的影响使用细胞计数试剂盒-8，集落形成试验，和流式细胞术。RT-qPCR和Westernblot检测RAF1的mRNA和蛋白表达。miR-605-3p与RAF1的3'-UTR的结合通过双荧光素酶报告基因测定来证实。
结果：我们的结果显示miR-605-3p在OS组织和细胞中显著降低。miR-605-3p水平较低与OS患者的淋巴结转移和5年总生存率较差密切相关。体外实验发现miR-605-3p抑制OS细胞增殖，促进细胞凋亡。机械上,原癌基因RAF1被视为miR-605-3p的靶标,在OS细胞中被miR-605-3p强烈抑制.RAF1的恢复明显消除了miR-605-3p对OS进展的抑制作用，提示RAF1是miR-605-3p的关键介质。与RAF1水平降低一致，miR-605-3p抑制OS细胞中MEK和ERK的激活，这是RAF1的目标。此外，在化疗耐药OS患者中发现较低水平的miR-605-3p，下调的miR-605-3p增加了OS细胞对治疗剂的抗性。
结论：我们的数据显示miR-605-3p通过调节RAF1和增加OS细胞的化学敏感性而成为肿瘤抑制基因，提供了miR-605-3p在OS中的新工作机制。工程化能够有效地将具有治疗活性的miR-605-3p递送到肿瘤中的稳定纳米囊泡可能是用于治疗OS的有希望的治疗方法。
目的：这项工作的目的是探讨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。
方法：通过逆转录定量PCR（RT-qPCR）评估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,miR-605-3p对OS细胞生长的影响使用细胞计数试剂盒-8，集落形成试验，和流式细胞术。RT-qPCR和Westernblot检测RAF1的mRNA和蛋白表达。miR-605-3p与RAF1的3'-UTR的结合通过双荧光素酶报告基因测定来证实。
结果：我们的结果显示miR-605-3p在OS组织和细胞中显著降低。miR-605-3p水平较低与OS患者的淋巴结转移和5年总生存率较差密切相关。体外实验发现miR-605-3p抑制OS细胞增殖，促进细胞凋亡。机械上,原癌基因RAF1被视为miR-605-3p的靶标,在OS细胞中被miR-605-3p强烈抑制.RAF1的恢复明显消除了miR-605-3p对OS进展的抑制作用，提示RAF1是miR-605-3p的关键介质。与RAF1水平降低一致，miR-605-3p抑制OS细胞中MEK和ERK的激活，这是RAF1的目标。此外，在化疗耐药OS患者中发现较低水平的miR-605-3p，下调的miR-605-3p增加了OS细胞对治疗剂的抗性。
结论：我们的数据显示miR-605-3p通过调节RAF1和增加OS细胞的化学敏感性而成为肿瘤抑制基因，提供了miR-605-3p在OS中的新工作机制。工程化能够有效地将具有治疗活性的miR-605-3p递送到肿瘤中的稳定纳米囊泡可能是用于治疗OS的有希望的治疗方法。
