国际肿瘤学杂志

国际肿瘤学杂志 ›› 2022, Vol. 49 ›› Issue (4): 193-198.doi: 10.3760/cma.j.cn371439-20220303-00034

• 论著 •    下一篇

miR-219a-5p通过负调控HMGA2抑制骨肉瘤U2OS细胞增殖、侵袭和迁移

周仁邦1, 张仲传2, 许志远1, 朱勋兵2()   

  1. 1蚌埠医学院组织移植安徽省重点实验室,蚌埠 233030
    2蚌埠医学院第二附属医院骨外科,蚌埠 233030
  • 收稿日期:2022-03-03 修回日期:2022-03-20 出版日期:2022-04-08 发布日期:2022-05-11
  • 通讯作者: 朱勋兵 E-mail:Zhuxb22@163

MiR-219a-5p inhibits the proliferation, invasion and migration of osteosarcoma U2OS cells by negatively regulating HMGA2

Zhou Renbang1, Zhang Zhongchuan2, Xu Zhiyuan1, Zhu Xunbing2()   

  1. 1Anhui Key Laboratory of Tissue Transplantation, Bengbu Medical College, Bengbu 233030, China
    2Department of Orthopedics, Second Affiliated Hospital of Bengbu Medical College, Bengbu 233030, China
  • Received:2022-03-03 Revised:2022-03-20 Online:2022-04-08 Published:2022-05-11
  • Contact: Zhu Xunbing E-mail:Zhuxb22@163

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摘要：

目的 研究miR-219a-5p通过调控高迁移率蛋白A2（HMGA2）对骨肉瘤U2OS细胞增殖、侵袭和迁移的影响。方法 采用实时定量PCR检测骨肉瘤U2OS细胞和正常成骨细胞hFOB1.19的miR-219a-5p mRNA表达水平。采用脂质体转染法将miR-219a-5p模拟物miR-219a-5p mimic（miR-219a-5p mimic组）以及阴性对照mimic NC（mimic NC组）转染U2OS细胞,通过实时定量PCR检测转染后细胞miR-219a-5p和HMGA2 mRNA的表达水平,蛋白质印迹法检测HMGA2蛋白水平,采用CCK-8法和克隆形成实验检测细胞增殖能力、划痕实验检测细胞迁移能力、Transwell小室侵袭实验检测细胞侵袭能力,利用双荧光素酶报告基因实验验证miR-219a-5p与HMGA2间的作用关系。结果 实时定量PCR结果显示,骨肉瘤U2OS细胞中的miR-219a-5p表达水平（0.11±0.01）显著低于正常成骨细胞（1.00±0.06）,差异具有统计学意义（t=26.83,P<0.001）。CCK-8法结果显示,mimic NC组和miR-219a-5p mimic组培养24 h后细胞吸光度值分别为0.52±0.02、0.42±0.02,48 h后分别为0.85±0.03、0.60±0.03,72 h后分别为1.12±0.02、0.72±0.02,miR-219a-5p mimic组细胞增殖活性显著低于mimic NC组,差异均具有统计学意义（t=6.97,P<0.001;t=16.65,P<0.001;t=26.78,P<0.001）。克隆形成实验结果显示,miR-219a-5p mimic组细胞克隆数为（157.00±15.39）个,明显低于mimic NC组的（294.00±15.51）个,差异具有统计学意义（t=9.70,P<0.001）。划痕实验结果显示,培养24 h后,miR-219a-5p mimic组细胞划痕面积百分比为（40.53±2.92）％,显著高于mimic NC组的（21.71±3.11）％,差异具有统计学意义（t=7.26,P=0.002）。Transwell小室侵袭实验结果显示,miR-219a-5p mimic组细胞的穿膜数量为（128.67±18.67）个,显著少于mimic NC组的（317.67±14.33）个,差异具有统计学意义（t=15.65,P<0.001）。双荧光素酶报告基因实验结果显示,在含有MUT-HMGA2细胞中,与mimic NC组（4.40±0.28）相比,转染miR-219a-5p mimic（4.30±0.26）对荧光素酶活性无明显影响,差异无统计学意义（t=0.85,P=0.690）。在含有WT-HMGA2细胞中,相比于NC mimic组（4.50±0.25）,miR-219a-5p mimic组（2.88±0.16）荧光素酶活性显著降低,差异具有统计学意义（t=19.15,P<0.001）。且过表达miR-219a-5p后,骨肉瘤U2OS细胞中的HMGA2 mRNA和蛋白表达（0.77±0.01;0.37±0.01）相比于mimic NC组（1.00±0.02;1.00±0.01）均下调,差异均具有统计学意义（t=16.38,P<0.001;t=42.02,P<0.001）。结论 在骨肉瘤细胞中,miR-219a-5p可通过下调HMGA2的表达抑制骨肉瘤细胞的增殖、迁移和侵袭。

关键词: 骨肉瘤, 微RNAs, HMGA2蛋白, 细胞增殖

Abstract:

Objective To investigate the effects of miR-219a-5p on proliferation, invasion and migration of osteosarcoma U2OS cells by regulating high mobility group A2 （HMGA2）. Methods Real-time quantitative PCR was used to detect miR-219a-5p mRNA expression levels in osteosarcoma U2OS cells and normal osteoblasts hFOB1.19. The U2OS cells were transfected with miR-219a-5p mimic （miR-219a-5p mimic group） and negative control mimic （mimic NC group） by liposome transfection. The expression levels of miR-219a-5p and HMGA2 mRNA in transfected cells were detected by real-time quantitative PCR. The level of HMGA2 protein was detected by Western blotting, cell proliferation ability was detected by CCK-8 assay and clonogenesis assay, cell migration ability was detected by scratching assay, cell invasion ability was detected by Transwell chamber assay, and the relationship between miR-219a-5p and HMGA2 was verified by double luciferase reporter gene assay. Results Real-time quantitative PCR showed that the expression level of miR-219a-5p in osteosarcoma U2OS cells （0.11±0.01） was significantly lower than that in normal osteoblasts （1.00±0.06）, with a statistically significant difference （t=26.83, P<0.001）. The results of CCK-8 showed that the cell absorbance values of the mimic NC group and miR-219a-5p mimic group were 0.52±0.02 and 0.42±0.02 after 24 h, 0.85±0.03 and 0.60±0.03 after 48 h, and 1.12±0.02 and 0.72±0.02 after 72 h respectively. The proliferation activity of the miR-219a-5p mimic group was significantly lower than that of the mimic NC group, with statistically significant differences （t=6.97, P<0.001; t=16.65, P<0.001; t=26.78, P<0.001）. The results of clonogenesis assay showed that the number of clones in the miR-219a-5p mimic group was 157.00±15.39, which was significantly lower than that in the mimic NC group （294.00±15.51）, with a statistically significant difference （t=9.70, P<0.001）. The results of scratch experiment showed that the percentage of scratch area in the miR-219a-5p mimic group was （40.53±2.92）% after 24 h culture, which was significantly higher than that in the mimic NC group [（21.71±3.11）%], with a statistically significant difference （t=7.26, P=0.002）. The results of Transwell chamber assay showed that the number of cells penetrating the membrane in the miR-219a-5p mimic group was 128.67±18.67, which was significantly lower than that in the mimic NC group （317.67±14.33）, with a statistically significant difference （t=15.65, P<0.001）. The results of double luciferase reporter gene assay showed that in MUT-HMGA2 cells, transfection with miR-219a-5p mimic （4.30±0.26） had no significant effect on luciferase activity compared with the mimic NC group （4.40±0.28）, with a statistically significant difference （t=0.85, P=0.690）. In WT-HMGA2 cells, compared with the mimic NC group （4.50±0.25）, the lucifase activity of the miR-219a-5p mimic group （2.88±0.16） was significantly decreased, with a statistically significant difference （t=19.15, P<0.001）. After miR-219a-5p was overexpressed, HMGA2 mRNA and protein expressions in osteosarcoma U2OS cells （0.77±0.01; 0.37±0.01） were downregulated compared with the mimic NC group （1.00±0.02; 1.00±0.01）, with statistically significant differences （t=16.38, P<0.001; t=42.02, P<0.001）. Conclusion In osteosarcoma cells, miR-219a-5p can inhibit the proliferation, migration and invasion of osteosarcoma cells by down regulating the expression of HMGA2.

Key words: Osteosarcoma, MicroRNAs, HMGA2 protein, Cell proliferation