miR-499a-5p在骨肉瘤中的表达及其对骨肉瘤发生发展的影响

Expression of miR-499a-5p in osteosarcoma and its effect on the occurrence and development of osteosarcoma

目的:探讨miR-499a-5p在骨肉瘤(OS)中的表达及其对OS发生发展的影响。方法:通过实时荧光定量PCR(RT-qPCR)验证miR-499a-5p在OS细胞系(143B、MNNG)及正常成骨细胞(hFOB1.19)中的差异表达,并使用3种在线预测工具对miR-499a-5p的靶基因进行了预测,并取3种数据共同交集得到的靶基因作为候选靶基因。利用靶基因进行基因功能分析(GO)富集分析,并利用京都基因与基因组百科全书(KEGG)通路富集分析,然后经过蛋白互作网络(PPI)分析,筛选节点最高的3个关键hub基因,并利用生存分析从这3个关键hub基因中筛选出具有预后意义的关键基因。结果:RT-qPCR结果显示,与正常的成骨细胞比较,OS细胞中miR-499a-5p表达降低(P<0.05)。通过3种在线预测工具预测miR-499a-5p的靶基因并交集获得181个靶基因。GO分析结果表明,生物过程(BP)最显著富集的是RNA聚合酶Ⅱ促子转录的正向调控,细胞组成(CC)最显著富集的是细胞核,分子功能(MF)最显著富集的是蛋白结合。在KEGG的通路富集中,最显著的是人类T细胞白血病病毒1感染信号通路,其次是轴突引导通路、催产素信号通路和T细胞激活信号通路。靶基因的蛋白互作网络分析发现节点最高的3个关键hub基因分别是异质核糖核蛋白C(HNRNPC)、真核翻译起始因子4γ2(EIF4G2)和真核翻译起始因子4A同工型2(EIF4A2)。生存分析结果显示,高度表达HNRNPC的OS患者的生存率显著降低(P<0.05)。结论:miR-499a-5p可能通过调控HNRNPC基因对OS发展产生潜在的影响。

Objective:To explore the expression of miR-499a-5p in osteosarcoma(OS) and its effect on the occurrence and development of OS.Methods:The differential expression of miR-499a-5p in OS cell line(143B,MNNG) and normal osteoblast(hFOB1.19) was verified by real-time fluorescence quantitative PCR(RT-qPCR).The target genes of miR-499a-5p were predicted by three on-line prediction tools,and the target genes obtained by the common intersection of three kinds of data were selected as candidate target genes.The target genes were used for Gene Ontology(GO) enrichment analysis.Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis and protein-protein interaction(PPI) network were used to screen the three key hub genes with the highest node,and the survival analysis was used to screen the prognostic key genes from the three key hub genes.Results:The results of RT-qPCR showed that the expression of miR-499a-5p in osteosarcoma cells was lower than that in normal osteoblasts(P<0.05).Three online prediction tools were used to predict the target genes of miR-499a-5p and 181 target genes were obtained by intersection.The results of GO analysis showed that the most significant enrichment of biological process(BP) was the positive regulation of RNA polymerase II promoter transcription,the most significant enrichment of cell composition(CC) was the nucleus,and the most significant enrichment of molecular function(MF) was protein binding.The most significant pathway of KEGG is human T cell leukemia virus I infection signal pathway(HTLV-I infection),followed by axon guidance pathway,oxytocin signaling pathway and T cell receptor signaling pathway.PPI analysis of target genes showed that the three key hub genes with the highest node were heterogeneous nuclear ribonucleoprotein C(HNRNPC),eukaryotic translation initiation factor 4γ2(EIF4G2) and eukaryotic translation initiation factor 4A isoform 2(EIF4A2).Survival analysis showed that the survival rate of OS patients with high expression of HNRNPC was significantly decreased(P<0.05).Conclusion:miR-499a-5p may play a potential role on the development of osteosarcoma by regulating HNRNPC gene.