Department of Orthopedics and Traumatology, The First Hospital of Zibo City, Zibo, China.
Department of Spine Surgery, The Third Hospital of Jinan, Jinan, China.
J Cell Biochem. 2019 Apr;120(4):6420-6430. doi: 10.1002/jcb.27929. Epub 2018 Nov 28.
Osteosarcoma (OS) is one of the most aggressive bone tumors. MicroRNAs (miRNAs) have been found to implicate in the pathogenesis of different types of cancers, including OS. This study aimed to explore the roles of miR-338-3p in OS and investigate the underlying mechanism. Human OS cell lines (MG-63 and U2OS) and osteoblast (hFOB) cell line were used in the study. The expression levels of miR-338-3p, runt-related transcription factor 2 (RUNX2) and cyclin-dependent kinase 4 (CDK4) were altered by transient transfection and determined by quantitative real-time polymerase chain reaction/Western blot analysis. Cell viability, colony numbers, migration, and invasion, and apoptotic cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, transwell assay, and flow cytometry assay, respectively. Dual luciferase reporter assay was performed to identify the target gene of miR-338-3p. Western blot assay was carried to measure the protein expression levels involved in cell apoptosis, migration, and mitogen-activated protein kinases (MAPK) pathway. We found that the expression of miR-338-3p was downregulated in MG-63 cell and U2OS cells, compared with hFOB cells. MiR-338-3p suppression significantly increased cell viability and colony numbers, promoted cell migration, and invasion, but suppressed cell apoptosis in MG-63 and U2OS cells. Opposite results were observed in the miR-338-3p overexpression. Interestingly, RUNX2 and CDK4 were direct target genes of miR-338-3p. RUNX2 inhibition shared a similar effect of miR-338-3p mimic on MG-63 cells. Furthermore, miR-338-3p inhibited the activation of MAPK pathway in MG-63 cells. To conclude, these findings suggested that miR-338-3p functioned as a tumor suppressor in OS cells by targeting RUNX2 and CDK4, as well as inhibition of the MAPK pathway.
骨肉瘤（OS）是最具侵袭性的骨肿瘤之一。已经发现 microRNAs（miRNAs）参与了不同类型癌症的发病机制，包括骨肉瘤。本研究旨在探讨 miR-338-3p 在骨肉瘤中的作用，并研究其潜在机制。研究中使用了人骨肉瘤细胞系（MG-63 和 U2OS）和成骨细胞（hFOB）细胞系。通过瞬时转染改变 miR-338-3p、 runt 相关转录因子 2（RUNX2）和细胞周期蛋白依赖性激酶 4（CDK4）的表达水平，并通过定量实时聚合酶链反应/ Western blot 分析进行测定。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定法、集落形成测定法、Transwell 测定法和流式细胞术分别测定细胞活力、集落数、迁移和侵袭以及凋亡细胞。进行双荧光素酶报告基因测定以鉴定 miR-338-3p 的靶基因。通过 Western blot 测定法测量涉及细胞凋亡、迁移和丝裂原活化蛋白激酶（MAPK）途径的蛋白表达水平。我们发现，与 hFOB 细胞相比，miR-338-3p 在 MG-63 细胞和 U2OS 细胞中的表达下调。miR-338-3p 抑制显著增加细胞活力和集落数，促进细胞迁移和侵袭，但抑制 MG-63 和 U2OS 细胞中的细胞凋亡。miR-338-3p 过表达则观察到相反的结果。有趣的是，RUNX2 和 CDK4 是 miR-338-3p 的直接靶基因。RUNX2 抑制在 MG-63 细胞中与 miR-338-3p 模拟物具有相似的作用。此外，miR-338-3p 抑制了 MG-63 细胞中 MAPK 途径的激活。总之，这些发现表明，miR-338-3p 通过靶向 RUNX2 和 CDK4 以及抑制 MAPK 途径，在骨肉瘤细胞中发挥肿瘤抑制作用。
