miR-654-3p靶向GMFB抑制骨肉瘤细胞增殖的作用及机制研究

目的 探究miR-654-3p对骨肉瘤细胞增殖的影响,预测其靶基因并评估靶基因对骨肉瘤患者生存预后的干预情况。方法 选择GEO数据库中GSE70367数据集进行差异分析,筛选骨肉瘤细胞中异常表达的miRNA,通过RT-qPCR检测miR-654-3p在骨肉瘤细胞系143B、MG-63、SAOS2和HOS中的表达情况。通过TargetScan和miRmap数据库预测miR-654-3p的靶基因,采用双荧光素酶报告基因实验验证miR-654-3p与GMFB基因的结合。用CCK8实验和CFU实验分析miR-654-3p和GMFB对骨肉瘤细胞MG-63和HOS增殖的影响。下载TCGA数据库中肉瘤患者的GMFB表达数据和临床信息,通过R软件包绘制GMFB对生存预后影响的KM曲线以及预测肉瘤患者1年、3年和5年生存率的列线图。结果 与人正常成骨细胞hFOB1.19相比,骨肉瘤细胞系143B、MG-63、SAOS2和HOS中miR-654-3p的表达均明显降低（P＜0.05）。miR-654-3p与GMFB基因结合,并负调控GMFB的表达。miR-654-3p模拟物在体外明显抑制骨肉瘤细胞的增殖,而GMFB转染能够逆转抑制作用（P＜0.05）。GMFB高表达的骨肉瘤患者的总体生存率明显低于低表达患者（P＜0.05）,且通过列线图能够预测患者的1年、3年和5年生存率。结论 miR-654-3p通过靶向负调控GMFB基因表达,抑制骨肉瘤细胞的增殖,且GMFB高表达与骨肉瘤患者的预后呈负相关。

Effect and mechanism of miR-654-3p inhibiting osteosarcoma cell proliferation by targeting GMFB

Objective To investigate the effect of miR-654-3p on the proliferation of osteosarcoma cells, predict its target genes and assess the intervention of target genes on the survival prognosis of osteosarcoma patients.Methods The GSE70367 dataset in the GEO database was selected for differential analysis to screen for aberrantly expressed miRNAs in osteosarcoma cells, and the expression of miR-654-3p in osteosarcoma cell lines 143B, MG-63, SAOS2 and HOS was detected by RT-qPCR. The target genes of miR-654-3p were predicted by TargetScan and miRmap database, and the binding of miR-654-3p to GMFB gene was verified using dual luciferase reporter gene assay. The effects of miR-654-3p and GMFB on the proliferation of MG-63 and HOS in osteosarcoma cells were analyzed by CCK8 assay and CFU assay. GMFB expression data and clinical information of sarcoma patients in the TCGA database were downloaded, and KM curves of the effect of GMFB on survival prognosis and column plots predicting 1-year, 3-year, and 5-year survival of sarcoma patients were plotted by the R package.Results The expression of miR-654-3p was significantly lower in osteosarcoma cell lines 143B, MG-63, SAOS2 and HOS compared to human normal osteoblasts hFOB1.19 (P<0.05). miR-654-3p bound to the GMFB gene and negatively regulated the expression of GMFB. The miR-654-3p mimics significantly inhibited the proliferation of osteosarcoma cells in vitro, while GMFB transfection was able to reverse the inhibitory effect (P<0.05). Overall survival was significantly lower in sarcoma patients with high GMFB expression than in patients with low expression (P<0.05), and 1-year, 3-year, and 5-year survival rates could be predicted by the nomogram.Conclusion miR-654-3p inhibits the proliferation of osteosarcoma cells by targeting the negative regulation of GMFB gene expression, and high GMFB expression is negatively correlated with the prognosis of sarcoma patients.