南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (1): 68-75.doi: 10.12122/j.issn.1673-4254.2023.01.09

• • 上一篇    下一篇

miR-125b-5p通过负调控RAB3D基因表达抑制骨肉瘤细胞增殖与迁移

谈秋瑜，吴 旭，纪 方，吴少云，颜 亮   

  1. 皖南医学院活性生物大分子安徽省重点实验室，安徽 芜湖 241002；海军军医大学附属长海医院，上海 200433；芜湖医药卫生学校，安徽 芜湖 241000
  • 出版日期:2023-01-20 发布日期:2023-02-22

miR-125b-5p inhibits proliferation and migration of osteosarcoma cells by negatively regulating RAB3D expression

TAN Qiuyu, WU Xu, JI Fang, WU Shaoyun, YAN Liang   

  1. Provincial Key Laboratory of Biological Macro- molecules Research, Wannan Medical College, Wuhu 241002, China; Changhai Hospital Affiliated to Naval Military Medical University, Shanghai 200433, China; Wuhu Medical and Health School, Wuhu 241000, China
  • Online:2023-01-20 Published:2023-02-22

RichHTML

29

PDF

437

摘要： 目的 探讨 miR-125b-5p 调控 RAB3D 在骨肉瘤增殖与迁移中的作用机制。方法 通过 qRT-PCR 检测正常骨细胞hFOB1.19、骨肉瘤细胞系（MG63、HOS）中miR-125b-5p的表达水平。骨肉瘤细胞系中通过脂质体转染升高或降低miR-125b-5p的表达，通过EDU和Transwell实验检测骨肉瘤细胞增殖和迁移的变化；通过生物信息学软件预测miR-125b-5p可能调控的靶基因为RAB3D；通过Western blot检测正常骨细胞hFOB1.19、骨肉瘤细胞系（MG63、HOS）中RAB3D的表达水平；骨肉瘤细胞系中通过脂质体转染升高或降低miR-125b-5p的表达，通过Western blot及qRT-PCR实验检测骨肉瘤细胞中RAB3D mRNA及蛋白表达水平；并在骨肉瘤细胞中通过脂质体转染升高或降低RAB3D的表达，通过EDU和Transwell实验检测骨肉瘤增殖和迁移的变化；随后在骨肉瘤细胞中通过脂质体转染同时升高miR-125b-5p和RAB3D的表达量，通过EDU和Transwell实验检测骨肉瘤细胞增殖和迁移的变化。结果 qRT-PCR结果表明在骨肉瘤细胞（MG63、HOS）中miR-125b-5p的表达明显下调（P<0.05）。3种生物信息学软件预测RAB3D是miR-125b-5p可能调控的靶基因。qRT-PCR、Western blot结果显示，miR-125b-5p 表达量升高，骨肉瘤细胞（MG63、HOS）中 RAB3D 的蛋白表达量下降（P<0.05）；miR-125b-5p 表达量降低，骨肉瘤细胞（MG63、HOS）中RAB3D的蛋白表达量下降（P<0.05），而RAB3D的mRNA水平没有发生变化。细胞增殖及迁移实验结果显示，miR-125b-5p与RAB3D对细胞增殖及迁移的影响相反，而同时在骨肉瘤细胞内升高miR-125b-5p与RAB3D的表达量，RAB3D对骨肉瘤细胞增殖及迁移的影响在重新升高miR-125b-5p表达量时被阻断（P<0.05）。结论 miR-125b-5p在转录后水平通过调控RAB3D的表达抑制了骨肉瘤细胞的增殖和迁移。

关键词: miR-125b-5p, RAB3D, 骨肉瘤细胞, 细胞增殖, 细胞迁移

Abstract: Objective To investigate the inhibitory effect of miR-125b-5p on proliferation and migration of osteosarcoma and the role of RAB3D in mediating this effect. Methods The expression level of miR-125b-5p was detected by qRT-PCR in a normal bone cell line (hFOB1.19) and in two osteosarcoma OS cell lines (MG63 and HOS). A miR-125b- 5p mimic or inhibitor was transfected in the osteosarcoma cell lines via liposome and the changes in cell proliferation and migration were detected with EDU and Transwell experiments. Bioinformatic analysis was conducted for predicting the target gene of miR-125b-5p, and the expression level of RAB3D in hFOB1.19, MG63, and HOS cells was detected by Western blotting. In the two osteosarcoma cell lines transfected with miR-125b-5p mimic or inhibitor, the expression levels of RAB3D mRNA and protein in osteosarcoma cells were examined with qRT-PCR and Western blotting. The effects of RAB3D overexpression, RAB3D knockdown, or overexpression of both miR-125b- 5p and RAB3D on the proliferation and migration of cells were assessed using EDU and Transwell experiments. Results The two osteosarcoma cell lines had significantly lower expression levels of miR-125b-5p (P<0.05). Bioinformatic analysis predicted that RAB3D was a possible target gene regulated by miR-125b-5p. In osteosarcoma cells, overexpression of miR-125b-5p significantly lowered the expression of RAB3D protein (P<0.05); inhibiting miR-125b-5p expression significantly decreased RAB3D expression only at the protein level (P<0.05) without obviously affecting its mRNA level. Modulation of miR-125b-5p and RAB3D levels produced opposite effects on proliferation and migration of osteosarcoma cells, and in cells with overexpression of both miR-125b-5p and RAB3D, the effect of RAB3D on cell proliferation and migration was blocked by miR-125b-5p overexpression (P<0.05). Conclusion Overexpression of miR-125b-5p inhibits the proliferation and migration of osteosarcoma cells by regulating the expression of RAB3D at the post-transcriptional level.

Key words: miR-125b-5p, RAB3D, osteosarcoma cells, cell proliferation, cell migration