南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (10): 1562-1568.doi: 10.12122/j.issn.1673-4254.2021.10.16

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MiR-671-5p通过负向调控SMAD3抑制骨肉瘤细胞的迁移和侵袭

胡雅琼，梁 答，陈新璐，陈 琳，白 俊，李洪利，尹崇高，钟 伟   

  1. 潍坊医学院病理学教研室，附属医院关节外一科//矫形骨科，医学研究实验中心，护理学院，山东 潍坊 261053
  • 出版日期:2021-10-20 发布日期:2021-11-11

MiR-671-5p negatively regulates SMAD3 to inhibit migration and invasion of osteosarcoma cells

HU Yaqiong, LIANG Da, CHEN Xinlu, CHEN Lin, BAI Jun, LI Hongli, YIN Chonggao, ZHONG Wei   

  1. Department of Pathology, First Department of Joint Surgery, Affiliated Hospital of Weifang Medical College, Medicine Research Center, College of Nursing, Weifang Medical University, Weifang 261053, China
  • Online:2021-10-20 Published:2021-11-11

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摘要： 目的 探讨miR-671-5p影响骨肉瘤的迁移侵袭的相关机制。方法 通过NCBI在线数据库筛选骨肉瘤中差异表达的微小RNA（miRNA）、预测miRNA的靶蛋白并进行功能分析；实时荧光定量PCR（qRT-PCR）用于检测转染过表达miR-671-5p质粒后con组和miR-671-5p组骨肉瘤细胞中miR-671-5p的表达情况；通过Transwell实验检测转染质粒后骨肉瘤细胞的迁移和侵袭能力；Western blot实验检测相关蛋白在骨肉瘤细胞中的表达情况；荧光素酶报告基因检测重组人SMAD家族成员3（SMAD3）的 3’UTR中是否含有miR-671-5p的结合位点。结果 MiR-671-5p在骨肉瘤组织和细胞中表达下调（P<0.05）。qRT-PCR证明转染过表达miR-671-5p质粒后，细胞转染成功（P<0.05）。过表达骨肉瘤细胞中的miR-671-5p后细胞的迁移和侵袭能力明显降低（P<0.05），并且miR-671-5p能够抑制骨肉瘤细胞的上皮间质转化（EMT）（P<0.05）；Western blot结果显示SMAD3在骨肉瘤细胞中表达上调（P<0.05）；荧光素酶报告基因检测证实miR-671-5p与SMAD3的3’UTR之间存在结合位点（P<0.05）；Western blot结果显示转染过表达SMAD3质粒后，SMAD3表达明显升高（P<0.05），而miR-671-5p能明显抑制SMAD3的表达（P< 0.05）；Transwell实验证明SMAD3能够促进骨肉瘤细胞的迁移和侵袭能力（P<0.05），而miR-671-5p则能够逆转这种促进作用（P<0.05）。结论 MiR-671-5p能够通过负向调控SMAD3抑制骨肉瘤细胞迁移和侵袭能力。

关键词: miR-671-5p, SMAD3, 迁移, 侵袭, 上皮间质转化, 骨肉瘤

Abstract: Objective To explore the role of miR-671-5p in regulating the migration and invasion of osteosarcoma and the underlying mechanisms. Methods The differentially expressed microRNAs (miRNAs) in osteosarcoma were screened in the NCBI online database, and the target proteins of these miRNAs were predicted and their functions were analyzed. Osteosarcoma cells were transfected with a plasmid overexpressing miR-671-5p, and the transfection efficiency was assessed using quantitative real-time PCR (qRT-PCR). The changes in the migration and invasion of the transfected cells were examined with Transwell assay, and the expressions of proteins related with epithelial-mesenchymal transition (EMT) were detected using Western blotting. Dual-luciferase reporter assay was performed to determine whether the 3'UTR of SMAD3 contained a targeted binding site of miR-671-5p. Results MiR-671-5p was significantly down-regulated in both osteosarcoma tissues and osteosarcoma cells (P<0.05). The osteosarcoma cells overexpressing miR-671-5p showed significantly reduced migration and invasion abilities (P<0.05) with obviously lowered expressions of EMT-related proteins (P<0.05). SMAD3 was highly expressed in osteosarcoma cells (P<0.05), and dual-luciferase reporter assay confirmed the presence of a targeted binding site between miR-671-5p and the 3'UTR of SMAD3 (P<0.05). In osteosarcoma cells transfected with a SMAD3-overexpressing plasmid (P<0.05), the high expression of SMAD3 significantly inhibited by miR-671-5p overexpression (P<0.05). Transwell assay demonstrated that SMAD3 overexpression significantly promoted the migration and invasion of osteosarcoma cells (P<0.05), and while miR-671-5p overexpression obviously reversed this effect (P<0.05). Conclusion MiR-671-5p can inhibit the invasion and migration of osteosarcoma cells by negatively regulating SMAD3.

Key words: miR-671-5p, SMAD3, migration, invasion, epithelial-mesenchymal transition, osteosarcoma