南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (2): 285-291.doi: 10.12122/j.issn.1673-4254.2021.02.18

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miR-300 通过负向调控垂体肿瘤转化基因 1 抑制骨肉瘤细胞MG63的侵袭和转移

梁 答, 吴晓林, 白 俊, 张丽萍, 尹崇高, 钟 伟   

  • 发布日期:2021-02-05

MiR-300 inhibits invasion and metastasis of osteosarcoma cell MG63 by negatively regulating PTTG1

  • Published:2021-02-05

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摘要：

目的 研究miR-300及垂体肿瘤转化基因1（PTTG1）对骨肉瘤侵袭转移的影响，探讨引起骨肉瘤侵袭转移的分子机制。方法 Western blot检测人成骨细胞hFOB1.19及骨肉瘤细胞MG63中PTTG1的表达情况，并检测转染敲低PTTG1质粒后细胞的转染效率；Transwell侵袭实验和CCK8实验检测敲低PTTG1及过表达miR-300对骨肉瘤细胞MG63侵袭和增殖能力的影响；网上预测并筛选与PTTG1互补结合的microRNAs（miRNAs）；qRT-PCR检测人成骨细胞hFOB1.19及骨肉瘤细胞MG63中miR-300的表达情况；Western blot检测转染过表达miR-300质粒后PTTG1在骨肉瘤细胞MG63的表达情况；双荧光素酶实验检测miR-300和PTTG1的靶向结合情况；Transwell侵袭实验和CCK8实验检测过表达miR-300和过表达PTTG1质粒共转后对骨肉瘤细胞MG63侵袭和增殖能力的影响。结果 PTTG1在骨肉瘤细胞MG63中表达较高（P=0.0002）；敲低PTTG1可抑制骨肉瘤细胞MG63的侵袭（P=0.0002）和增殖（P=0.0039）能力；网上预测软件预测可能与PTTG1互补结合的miRNAs，NCBI数据库下载数据集分析确定研究对象为miR-300；qRT-PCR结果表明miR-300在骨肉瘤细胞MG63中表达降低（P=0.0004）；采用过表达质粒过表达骨肉瘤细胞MG63中的miR-300，qRT-PCR结果提示转染成功（P<0.0001）；Western blot发现过表达miR-300后PTTG1的表达明显降低（P=0.0007）；双荧光素酶实验结果显示miR-300可与PTTG1靶向结合（P=0.0010）；细胞共转实验显示过表达PTTG1可逆转miR-300对骨肉瘤细胞侵袭（P=0.0003）和增殖（P=0.0077）能力的影响。结论 miR-300通过靶向结合PTTG1抑制骨肉瘤细胞MG63的侵袭转移能力。

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Abstract:

Objective To investigate the effects of miR-300 and PTTG1 on osteosarcoma invasion and metastasis and explore the molecular mechanism of osteosarcoma invasion and metastasis. Methods Western blot was used to detect the expression of PTTG1 in human osteoblasts hFOB1.19 and osteosarcoma cell MG63 and to detect the transfection efficiency of cells transfected with PTTG1-knockdown plasmid; Transwell invasion assay and CCK8 assay detected the effects of knockdown of PTTG1 and overexpression of miR-300 on the invasion and proliferation of osteosarcoma cell MG63. On-line prediction and screening of microRNAs (miRNAs) with complementary PTTG1 binding was conducted. qRT-PCR was performed to examine the expression of miR-300 in hFOB1.19 and MG63 cells, and Western blotting was used to detect the expression of PTTG1 in MG63 cells after transfection with a miR- 300 plasmid. Double luciferase assay was used to detect the targeted binding of miR-300 and PTTG1, Transwell invasion assay and CCK8 assay were used to detect the effects of overexpression of miR-300 and overexpression of PTTG1 plasmid on invasion and proliferation of osteosarcoma cell line MG63. Results PTTG1 was highly expressed in MG63 cells (P=0.0002). PTTG1 knockdown significantly inhibited the invasion (P=0.0002) and proliferation (P=0.0039) of MG63 cells. Based on the results of online prediction of complementary miRNAs to PTTG1 and analysis of the data from NCBI database, miR-300 was determined as the target miRNA in this study. qRT-PCR results showed a significantly decreased expression of miR-300 in MG63 cells (P=0.0004). Overexpression of MiR-300 in MG63 cells significantly decreased the expression of PTTG1 (P=0.0007), and the expressions of miR-300 and PTTG1 were negatively correlated. Dual luciferase assay showed that miR-300 could specifically bind to PTTG1 (P=0.001). Overexpression of PTTG1 could significantly reverse the effect of miR-300 overexpression on invasion (P=0.0003) and proliferation (P=0.0077) of MG63 cells. Conclusion Overexpression of miR-300 can inhibit the invasion and metastasis of osteosarcoma cell MG63 by targeting PTTG1.

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