氧化苦参碱调控肝细胞癌进展枢纽基因的筛选及其作用机制

Screening of hub genes of oxymatrine regulating HCC progression and its mechanism

目的基于生物信息学方法筛选氧化苦参碱调控肝细胞癌(HCC)进展的枢纽基因,并分析其circ_0031450相关的内源竞争RNA(ceRNA)机制。方法通过circBANK、miRMAP及miRWALK等多个数据库预测并下载整理circ_0031450的miRNA靶点,利用Cytoscape软件构建ceRNA网络。通过STRING网站构建蛋白—蛋白互作(PPI)网络文件,并导入Cytoscape软件进行可视化,利用MCODE插件筛选关键基因并创建其子网络。利用R软件基于CIBERSORT算法,筛选免疫浸润相关性最显著的基因LCK作为枢纽基因。对LCK进行免疫检查点相关性分析及基因集富集分析(GSEA分析);通过GEPIA2数据库预测与LCK相关的前150个基因,通过R软件对其进行基因本体论(GO)及京都基因与基因组百科全书(KEGG)富集分析;比较LCK高、低表达组的总生存期(OS),对氧化苦参碱及LCK进行分子对接。结果共得到3个miRNA(miR-548c-5p、miR-548d-5p及miR-648)作为circ_0031450的靶点,得到28个mRNA作为miRNA及氧化苦参碱的靶点,并成功构建ceRNA网络、PPI网络及关键基因子网络。LCK表达与HCC免疫微环境中多个免疫细胞及免疫检查点的表达具有相关性(P均<0.05)。GSEA分析及GO、KEGG富集分析结果显示,LCK主要参与T、B淋巴细胞的激活、分化及其受体信号、免疫应答、细胞因子相互作用及趋化因子信号等生物学过程或通路。LCK高表达组OS高于LCK低表达组(P<0.05),氧化苦参碱与LCK具有良好的结合活性。结论氧化苦参碱可能通过作用于LCK并间接调控circ_0031450相关ceRNA机制,参与改变HCC的免疫微环境,从而影响HCC的发生与进展。

Objective To screen the hub genes involved in the regulation of oxymatrine on hepatocellular carcinoma(HCC)progression by bioinformatics method and to analyze the endogenous competitive RNA(ceRNA)mechanism associated with circ_0031450.Methods The miRNA targets of circ_0031450 were predicted and downloaded from circ‑BANK,miRMAP,and miRWALK databases,and Cytoscape was used to construct the ceRNA network.We constructed the protein-protein interaction(PPI)network files via the STRING website and imported Cytoscape software for visualization,screened essential genes,and created subnetworks using the MCODE plug-in.Based on the CIBERSORT algorithm,R software was used to filter the most significant gene of immune infiltration as the hub gene.Immunological checkpoint correlation analysis and gene set enrichment analysis(GSEA)were performed for LCK.The top 150 genes related to LCK were predicted by the GEPIA2 database,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed by R software.The overall survival time(OS)of high and low LCK expression groups was compared,and molecular docking was performed between oxymatrine and LCK.Results Three miRNAs(miR-548c-5p,miR-548d-5p,and miR-648)were obtained as the targets of circ_0031450,and 28 mRNAs were ob-tained as the targets of miRNAs and oxymatrine.The ceRNA network,PPI network,and key gene subnetwork were con-structed successfully.LCK expression was significantly correlated with the expression of multiple immune cells and im-mune checkpoints in the HCC immune microenvironment.The results of GSEA analysis and GO and KEGG enrichment analysis showed that LCK was mainly involved in T and B lymphocyte activation,differentiation,receptor signal,immune response,cytokine interaction,chemokine signal,and other biological processes or pathways.OS in the high-expression group was significantly higher than in the low-expression group(P<0.05),and oxymatrine had a good binding activity with LCK.Conclusion Oxymatrine may be involved in changing the immune microenvironment of HCC by acting on LCK and indirectly regulating circ_0031450-related ceRNA mechanism,thus affecting the development and progression of HCC.