基于高通量测序探讨原发性肝癌预后标志物及中药靶向预测研究

Screening of prognostic markers for primary liver cancer and predictive analysis of potential traditional Chinese medicine based on RNA-seq

目的:利用高通量测序技术筛选原发性肝癌(PLC)的预后标志物,并预测其潜在靶向中药。方法:纳入PLC患者103例及健康人50名,采集外周血并提取外周血单个核细胞(PBMC)进行高通量测序。利用生物信息学分析技术对基因表达矩阵进行差异分析,Cytoscape筛选核心基因,“CIBERSORT”反卷积算法和单样本基因集富集分析进行免疫浸润评分,“KM-plot”绘制生存曲线及Coremine Medical数据库获取靶向中药。结果:相较于健康对照组,404个基因在PLC患者PBMC中差异表达,主要参与气体运输、snRNP复合物形成、mRNA剪接等生物过程,在尼古丁成瘾、酗酒、溶酶体、花生四烯酸代谢等通路富集。9个核心基因包括SLC4A1、ALAS2、HBG1、AHSP、HBG2、HBB、HBD、HBA2和HBA1,其中HBA2、AHSP和HBG2高表达与PLC患者较短的生存期相关。静息态NK细胞、CD8^(+)T细胞、幼稚B淋巴细胞和活化记忆CD4^(+)T细胞等正免疫调控细胞群在PLC患者PBMC中占比减少,而抑制性免疫细胞群包括单核细胞、M0型巨噬细胞及调节性T淋巴细胞等浸润增加。生存分析显示静息态NK细胞浸润水平与PLC患者总体生存率呈正相关。此外,腹腔积液、肝功能Child-pugh评分及BCLC分期也是量化PLC预后的重要参考指标。鳖甲、阿胶、荷叶、茶树根、地龙在内的30味中药可能参与PLC靶向治疗。结论:本研究为PLC预后标志的鉴定和潜在靶向中药研究提供新方向。

Objective:To identify prognostic markers for primary liver cancer(PLC) through high-throughput sequencing technology and predict its potential targeted traditional Chinese medicines(TCMs).Methods:A total of 103 patients with PLC and 50 healthy controls were recruited.Peripheral blood samples were collected,and peripheral blood mononuclear cell(PBMC)were isolated for subsequent high-throughput sequencing analysis.The gene expression matrix was analyzed using bioinformatics analysis technology,core genes were screened using Cytoscape,immune infiltration was scored using the ‘CIBERSORT'deconvolution algorithm and single-sample gene set enrichment analysis,survival curve was plotted using KM-plot,and targeted TCMs was obtained from the Coremine Medical database.Results:A total of 404 genes were found to be differentially expressed in PLC group.It is mainly involved in biological processes such as gas transport,snRNP complex formation,and mRNA splicing,and is enriched in nicotine addiction,alcoholism,lysosome and arachidonic acid metabolism.Among the nine core genes,namely SLC4A1,ALAS2,HBG1,AHSP,HBG2,HBB,HBD,HBA2 and HBA1.It was observed that higher expression levels of HBA2,AHSP and HBG2 were significantly associated with reduced survival time in patients with PLC.The proportion of positive immune regulatory cell subsets,such as quiescent natural killer(NK) cells,CD8^(+) T cells,naive B lymphocytes,and activated memory CD4^(+) T cells in PBMC of patients with PLC,is diminished.Meanwhile,there is an increased infiltration of inhibitory immune cell subsets including monocytes,M0 macrophages,and regulatory T lymphocytes.Survival analysis showed that the level of NK cell infiltration at resting-state was positively correlated with the overall survival of PLC patients.Furthermore,ascites,Child-Pugh score,and BCLC stage serve as crucial prognostic indicators for quantifying the prognosis of PLC.Thirty kinds of TCMs,including turtle shell,donkey-hide gelatin,lotus leaf,tea tree root,and ground swallowwort,may be involved in the targeted therapy of PLC.Conclusion:This study offers a new avenue for identifying prognostic markers and conducting research on TCMs.