miRNA-1-3p通过肌细胞增强因子2A对骨肉瘤细胞生物学功能的影响

Effect of miRNA-1-3p on biological function of osteosarcoma cells via myocyte enhancer factor 2A

目的探讨miRNA-1-3p(miR-1-3p)对骨肉瘤细胞肌细胞增强因子2A(MEF2A)表达及生物学功能的影响。方法收集2019年1月至2020年1月山西省肿瘤医院临床确诊的20例骨肉瘤患者的肿瘤组织及癌旁正常组织,使用实时荧光定量聚合酶链反应(qRT-PCR)检测样本中miR-1-3p表达水平。采用qRT-PCR检测骨肉瘤细胞株U2-OS、SAOS-2、MG63、SW1353及人正常成骨细胞株hFOB1.19中miR-1-3p表达水平,选择miR-1-3p表达水平最低的细胞株进行后续实验。构建过表达miR-1-3p载体(miR-1-3p mimcs),转染miR-1-3p mimcs的细胞为miR-1-3p过表达组,转染空载体(miR-1-3p nc)的细胞为对照组;使用CCK-8法检测细胞增殖活性,流式细胞术检测细胞凋亡和细胞周期变化。采用miRwalk网站工具预测miR-1-3p靶基因,并通过双荧光素酶报告基因实验进行验证;蛋白质印迹法检测各组细胞MEF2A蛋白的表达。结果与癌旁组织相比,骨肉瘤组织中miR-1-3p表达下调(0.31±0.14比0.62±0.21),差异有统计学意义(t=5.31,P<0.01)。miR-1-3p在骨肉瘤U2-OS细胞中表达最低;与对照组相比,miR-1-3p过表达组细胞U2-OS细胞增殖活性受抑制(48 h吸光度值0.56±0.01比0.77±0.03,t=2.77,P<0.01;72 h吸光度值0.87±0.02比1.40±0.03,t=2.93,P<0.01),细胞周期G1至S期阻滞增加[G1期(38.24±0.55)%比(32.11±0.80)%,t=9.27,P=0.01;S期(61.24±0.90)%比(67.78±0.83)%,t=7.52,P=0.02],细胞早期凋亡率升高[(11.20±0.12)%比(1.50±0.12)%,t=2.91,P<0.05]。miRwalk网站工具预测miR-1-3p靶基因为MEF2A。双荧光素酶报告基因检测结果显示,miR-1-3p和MEF2A 3’UTR靶向结合,miR-1-3p过表达组U2-OS细胞荧光素酶活性低于对照组(海肾荧光素酶与萤火虫荧光素酶活性比值0.53±0.06比1.00±0.04,t=4.04,P<0.05);蛋白质印迹法结果显示,miR-1-3p过表达组U2-OS细胞MEF2A蛋白表达水平较对照组低(蛋白相对表达量0.41±0.14比0.77±0.12,t=3.93,P<0.05)。结论 miR-1-3p低表达可能与骨肉瘤细胞增殖、凋亡和周期改变相关。miR-1-3p可负向调控MEF2A蛋白表达并调控骨肉瘤的发生、发展。

Objective To investigate the effect of miRNA-1-3p(miR-1-3p)on expression of myocyte enhancer factor 2A(MEF2A)and the biological function of osteosarcoma cells.Methods The tumor tissues and adjacent normal tissues of 20 patients with osteosarcoma who were clinically diagnosed in Shanxi Provincial Cancer Hospital from January 2019 to January 2020 were collected,and the expression of miR-1-3p in the samples was detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).The expression of miR-1-3p in osteosarcoma cell lines U2-OS,SAOS-2,MG63,SW1353 and human normal osteoblast cell line hFOB1.19 was detected by qRT-PCR,then the cell line with the lowest expression of miR-1-3p was selected for follow-up experiments.An overexpression miR-1-3p vector was constructed(miR-1-3p mimcs).The miR-1-3p overexpression group was transfected with miR-1-3p mimcs,and the control group was transfected with empty vector(miR-1-3p nc).CCK-8 method was used to detect the proliferation activity of cells;flow cytometry was used to detect the changes of cell apoptosis and cell cycle.miRwalk database was used to predict the miR-1-3p target gene,and the target gene was verified by dual-luciferase reporter gene assay;Western blot was used to detect the expression of MEF2A protein in cells of each group.Results Compared with adjacent tissues,the expression of miR-1-3p in osteosarcoma tissues was down-regulated(0.31±0.14 vs.0.62±0.21),and the difference was statistically significant(t=5.31,P<0.01).The expression of miR-1-3p in U2-OS cells was the lowest;compared with the control group,the proliferation activity of U2-OS cells was inhibited in miR-1-3p overexpression group(48 h absorbance value 0.56±0.01 vs.0.77±0.03,t=2.77,P<0.01;72 h absorbance value 0.87±0.02 vs.1.40±0.03,t=2.93,P<0.01);G1/S cell cycle arrest increased[G1 phase(38.24±0.55)%vs.(32.11±0.80)%,t=9.27,P=0.01;S phase(61.24±0.90)%vs.(67.78±0.83)%,t=7.52,P=0.02];early apoptotic rate increased[(11.20±0.12)%vs.(1.50±0.12)%,t=2.91,P<0.05],miRwalk database predicted that the miR-1-3p target gene was MEF2A.The result of dual-luciferase reporter gene assay showed that miR-1-3p bound to MEF2A 3'UTR,and the luciferase activity of U2-OS cells in miR-1-3p overexpression group was lower than that in the control group(renilla luciferase/firefly luciferase activity ratio 0.53±0.06 vs.1.00±0.04,t=4.04,P<0.05).Western blot showed that the expression of MEF2A protein in U2-OS cells of miR-1-3p overexpression group was lower than that of the control group(protein relative expression 0.41±0.14 vs.0.77±0.12,t=3.93,P<0.05).Conclusions The low expression of miR-1-3p may be associated with the proliferation,apoptosis and cycle changes of osteosarcoma cells.miR-1-3p can negatively regulate the expression of MEF2A protein and regulate the occurrence and development of osteosarcoma.