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

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

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-cadhe-rin,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.

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.Results The 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.