环状RNA hsa_circ_0003568通过miR-140-3p及miR-502-5p调控靶基因CDK6及NFYA在骨肉瘤中的作用

The effect of circRNA hsa_circ_0003568 regulating target genes CDK6 and NFYA in osteosarcoma through miR-140-3p and miR-502-5p

目的探讨环状RNA hsa_circ_0003568在骨肉瘤(OS)中的表达并采用生物信息学方法进行circRNA-miRNA-mRNA的网络构建,分析其可能的机制及临床意义。方法采用实时荧光定量聚合酶链反应(RT-qPCR)检测hsa_circ_0003568在OS细胞系及正常成骨细胞中的表达。随后,基于circRNA-miRNA和miRNA-mRNA构建circRNA-miRNA-mRNA网络。通过多种circRNA软件数据库进行分析hsa_circ_0003568靶向的miRNA,并进一步通过miRNA数据软件预测其靶基因,选择至少4个以上软件支持的靶向miRNA及靶基因,并通过GEPIA数据库软件对靶基因进行生存分析筛选出显著预后差异的靶基因从而分析hsa_circ_0003568潜在的临床意义。结果RT-qPCR检测结果表明:与正常对照组相比,hsa_circ_0003568在OS细胞系中显著升高(P<0.05)。测序发现hsa_circ_0003568的成环连接点位于IST1前体RNA的第5和6外显子上。生物信息学分析结果显示:hsa_circ_0003568同时靶向两个miRNA(miR-140-3p及miR-502-5p),构建了hsa_circ_0003568、miR-140-3p/miR-502-5p及13个靶基因的circRNA-miRNA-mRNA网络,此外,GEPIA数据库分析中发现CDK6和NFYA两个靶基因,与OS患者的生存预后显著相关(P<0.05),并且NFYA同时是miR-140-3p及miR-502-5p的潜在靶基因。结论高表达的hsa_circ_0003568可能通过miR-140-3p及miR-502-5p调控基因CDK6及NFYA参与OS的发生发展过程。

Objective To investigate the expression of circRNA hsa_circ_0003568 in osteosarcoma(OS)and to construct circRNA-miRNA-mRNA network by means of bioinformatics methods.Methods Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of hsa_circ_0003568 in osteosarcoma cell lines and normal osteoblasts.Subsequently,a circRNA-miRNA-mRNA network was constructed based on circRNA-miRNA and miRNA-mRNA.Various circRNA software databases were used to analyze the miRNA targeted by hsa_circ_0003568,and its target genes were predicted by miRNA data software.Targeted miRNA and target genes supported by at least 4 software were selected.Target genes with significant prognostic difference were selected through survival analysis of target genes with the database software GEPIA,so as to analyze the potential clinical significance of hsa_circ_0003568.Results The RT-qPCR analysis showed that hsa_circ_0003568 was significantly increased in OS cell lines in comparison with that of the normal control group(P<0.05).Sequencing revealed that the ring-forming linkage point of hsa_circ_0003568 was located on the 5th and 6th exons of IST1 precursor RNA.The bioinformatics analysis showed that hsa_circ_0003568 simultaneously targeted two miRNAs(miR-140-3p and miR-502-5p)and constructed the circRNA-miRNA-mRNA network based on hsa_circ_0003568,miR-140-3p/miR-502-5p and 13 target genes.In addition,two target genes,CDK6 and NFYA,found in GEPIA database analysis,were significantly associated with survival prognosis in OS patients(P<0.05).Moreover,NFYA was the potential target gene of both miR-140-3p and miR-502-5p.Conclusion The highly expressed hsa_circ_0003568 may be involved in the occurrence and development of OS through the regulation of genes CDK6 and NFYA by miR-140-3p and miR-502-5p.