Deng Yi, Luan Fujun, Zeng Li, Zhang Yanjun, Ma Kunlong
Department of Oncology, Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, China.
Department of Orthopedics, Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, China.
EXCLI J. 2017 May 5;16:618-627. doi: 10.17179/excli2017-258. eCollection 2017.
MiR-429 functions as a tumor suppressor and has been observed in multiple types of cancer, but the effects and mechanisms of miR-429 in osteosarcoma are poorly understood. This study is performed to evaluate the functions of miR-429 in the progression of osteosarcoma. Firstly, the miR-429 expression in osteosarcoma tissues and osteosarcoma cells was detected using real time PCR, and the relationship between miR-429 expression and overall survival of osteosarcoma was analyzed. Secondly, the effects of miR-429 on the migration, invasion, proliferation and apoptosis of osteosarcoma cells were evaluated using transwell assay, wound-healing assay, CCK-8 assay and flow cytometry, respectively. Proteins related to epithelial-mesenchymal transition (EMT), E-cadherin, Vimentin, N-cadherin and Snail, were also detected using Western blot. Finally, the target gene of miR-429 in osteosarcoma was predicted and verified using dual luciferase assay and the expression correlation between them was analyzed using Pearson's correlation. MiR-429 was down-regulated in osteosarcoma tissues and osteosarcoma cells; the expression level of miR-429 was associated with the prognosis of osteosarcoma. High level of miR-429 in osteosarcoma cells significantly suppressed the migration, invasion and proliferation of cells but induced cells apoptosis. Furthermore, high level of miR-429 in osteosarcoma cells obviously increased the expression of E-cadherin protein but decreased the expression of Vimentin, N-Cadherin and Snail proteins. EMT inducer ZEB1 was the target gene of miR-429 and the expression of ZEB1 was negatively related to the miR-429 expression in osteosarcoma. In conclusion, miR-429 may functions as a tumor suppressor and be down-regulated in osteosarcoma. MiR-429 may suppress the progression and metastasis of osteosarcoma by down-regulating the ZEB1 expression.
微小RNA-429（miR-429）作为一种肿瘤抑制因子，已在多种癌症类型中被观察到，但其在骨肉瘤中的作用及机制尚不清楚。本研究旨在评估miR-429在骨肉瘤进展中的作用。首先，采用实时定量聚合酶链反应（real time PCR）检测骨肉瘤组织和骨肉瘤细胞中miR-429的表达，并分析miR-429表达与骨肉瘤总生存期的关系。其次，分别采用Transwell实验、划痕实验、CCK-8实验和流式细胞术评估miR-429对骨肉瘤细胞迁移、侵袭、增殖和凋亡的影响。同时，采用蛋白质免疫印迹法检测与上皮-间质转化（EMT）相关的蛋白质，如E-钙黏蛋白、波形蛋白、N-钙黏蛋白和蜗牛蛋白（Snail）。最后，采用双荧光素酶报告基因实验预测并验证miR-429在骨肉瘤中的靶基因，并使用Pearson相关性分析它们之间的表达相关性。miR-429在骨肉瘤组织和骨肉瘤细胞中表达下调；miR-429的表达水平与骨肉瘤的预后相关。骨肉瘤细胞中高水平的miR-429显著抑制细胞的迁移、侵袭和增殖，但诱导细胞凋亡。此外，骨肉瘤细胞中高水平的miR-429明显增加E-钙黏蛋白的表达，但降低波形蛋白、N-钙黏蛋白和蜗牛蛋白的表达。EMT诱导因子锌指蛋白E盒结合因子1（ZEB1）是miR-429的靶基因，且ZEB1的表达与骨肉瘤中miR-429的表达呈负相关。综上所述，miR-429可能作为一种肿瘤抑制因子，在骨肉瘤中表达下调。miR-429可能通过下调ZEB1的表达来抑制骨肉瘤的进展和转移。
