国际肿瘤学杂志

国际肿瘤学杂志 ›› 2017, Vol. 44 ›› Issue (7): 490-495.doi: 10.3760/cma.j.issn.1673-422X.2017.07.003

• 论著 • 上一篇    下一篇

微小RNA-24对骨肉瘤细胞U2OS体外增殖与迁移能力的影响及作用机制

贺启华   

  1. 723000 陕西省汉中市三二〇一医院肿瘤内三科
  • 收稿日期:2017-01-19 出版日期:2017-07-08 发布日期:2017-06-20
  • 通讯作者: 贺启华，Email: nyzhangpeihua@163.com E-mail:nyzhangpeihua@163.com

Effect and mechanism of microRNA24 on cell proliferation and migration of osteosarcoma cell line U2OS

He Qihua   

  1. hird Department of Medical Oncology, San Er Ling Yi Hospital of Hanzhong, Shaanxi Province, Hanzhong 723000, China
  • Received:2017-01-19 Online:2017-07-08 Published:2017-06-20
  • Contact: He Qihua E-mail:nyzhangpeihua@163.com

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摘要： 目的  探讨微小RNA-24（miR-24）对人骨肉瘤细胞株U2OS体外增殖与迁移能力的影响及其可能的机制。方法  通过转染miR-24模拟物（miR-24 mimic）建立miR-24过表达的U2OS细胞株，采用实时荧光定量逆转录聚合酶链反应（qRT-PCR）、细胞活力检测方法（CCK-8法）和Transwell检测对照组、阴性对照组和miR-24过表达组3组细胞增殖及迁移能力的变化； Western blotting检测细胞的上皮间质转化（EMT）进程及核转录因子-κB（NF-κB）信号通路的活性。结果  对照组、阴性对照组和miR-24过表达组miR-24的表达水平分别为1.00±0.00、1.03±0.08和2.46±0.29，3组间差异有统计学意义（F=11.026，P=0.012），转染了miR24 mimic后，人骨肉瘤细胞株U2OS中的miR-24表达水平与对照组相比显著升高（t=4.604，P=0.009）。与对照组相比，人骨肉瘤细胞株U2OS过表达miR-24 24 h［(3.56±0.27)%∶(8.63±0.79)%］、48 h［（20.16±1.09)%∶(54.77±5.42)%）］和72 h［(45.47±3.16)%∶(95.52±8.56)%］后细胞增长率均明显降低，差异均有统计学意义（t=3.896，P=0.016；t=4.813，P=0.008；t=7.173，P=0.002）。过表达miR-24 24 h后，对照组、阴性对照组和miR24过表达组细胞相对迁移率分别为(100.00±0.00)%、(99.26±5.85)%和(31.37±2.09)%，3组间差异有统计学意义（F=12.175，P=0.009）；与对照组相比，过表达miR-24后细胞迁移率明显降低（t=3.843， P=0.014）。同时过表达miR24上调细胞中上皮细胞标志物上皮细胞钙黏蛋白（E-cadherin，t=3.852，P=0.018）和β-连环素蛋白（β-catenin）的表达（t=3.512，P=0.024），而下调间质细胞标志物神经钙黏蛋白（N-cadherin，t=3.832，P=0.018）和波形蛋白（vimentin)的表达（t=4.058，P=0.012），抑制U2OS的EMT进程。此外，过表达miR24可下调NFκB(p65)（t=4.813，P=0.008）、磷酸化核因子IκB激酶抑制剂α（p-IKK-α）（t=3.764，P=0.013）和磷酸化激活IκB激酶复合物α（p-IκB-α)的表达（t=4.064，P=0.012），抑制NF-κB信号通路的激活。结论  miR-24可抑制人骨肉瘤细胞株U2OS体外增殖与迁移，其作用机制可能与抑制NF-κB信号通路的激活，进而抑制细胞的EMT进程相关。提示miR-24可作为治疗骨肉瘤迁移的一个潜在作用位点。

关键词: 骨肉瘤, 细胞运动, 微小RNA-24

Abstract: Objective  To investigate the effect of microRNA-24 (miR-24) on cell proliferation and migration in osteosarcoma cell line U2OS and its possible mechanism. Methods  U2OS cell line with miR-24 overexpression was established by transfecting miR-24 mimic, and then cell proliferation and migration in control group, negative control group and miR-24 over expression group were detected with realtime quantitative reverse transcriptase polymerase chain reaction (qRTPCR) assay, cell counting kit8 (CCK8) assay and Transwell assay, respectively. Epithelial mesenchymal transition (EMT) progression and activation of nuclear factorκB (NF-κB) pathway were detected by Western blotting. ResultsThe expressions of miR24 in the control group, negative control group and miR24 overexpression group were 1.00±0.00, 1.03±0.08 and 2.46±0.29, with significant difference (F=11.026, P=0.012). Compared with the control group, the expression of miR-24 in human osteosarcoma U2OS cell line was significantly increased after transfecting miR24 mimic (t=4.604, P=0.009). After overexpression of miR24 for 24 h, 48 h and 72 h, U2OS cell viabilities were decreased significantly compared with the control group ［(3.56±0.27)% vs. (8.63±0.79)%, t=3.896, P=0.016; (20.16±1.09)% vs. (54.77±5.42)%, t=4.813, P=0.008; (45.47±3.16)% vs. (95.52±8.56)%, t=7.173, P=0.002)］. After over expression of miR-24 for 24 h, the cell migration rates in control group, negative control group and miR24 overexpression group were (100.00±0.00)%, (99.26±5.85)% and (31.37±2.09)%, respectively, and there was statistically significant difference among the three groups (F=12.175, P=0.009); and compared with control group, cell migration rate was decreased significantly after overexpression of miR-24 (t=3.843, P=0.004). Meanwhile, overexpression of miR-24 upregulated the expression levels of epithelial cell markers E-cadherin (t=3.852, P=0.018) and β-catenin (t=3.512, P=0.024), while down regulated the expression levels of mesenchymal cell markers N-cadherin (t=3.832, P=0.018) and vimentin (t=4.058, P=0.012), with a suppressed EMT progress. Other than that, miR-24 over expression inhibited the expressions of NF-κB (p65) (t=4.813, P=0.008), phosphorylation inhibitor of nuclear factor kappaB kinase α (p-IKK-α) (t=3.764, P=0.013) and phosphorylation inhibitor of nuclear factor kappa-B kinase complex α (p-IκB-α) (t=4.064, P=0.012), suppressing the activation of NFκB pathway. Conclusion  miR-24 can suppress cell proliferation and migration of osteosarcoma cell line U2OS in vitro, and its mechanism may be related to the suppression of NF-κB activation and EMT progression. It hints that miR-24 may be used as a potential new target for osteosarcoma therapy.

Key words: Osteosarcoma, Cell movement, MicroRNA-24