林家茂 张晨月 部帅 李振祥 赵成龙 王海永

摘要 目的：筛选中药单体蟾毒灵促进人肝癌细胞凋亡的潜在分子靶点。方法：以人肝癌细胞株PLC/PRF/5为体外模型，观察中药单体蟾毒灵对人肝癌细胞凋亡以及相关基因表达的影响。以不同浓度的蟾毒灵分别作用于PLC/PRF/5细胞24 h、48 h、72 h，检测其对人肝癌细胞增殖和凋亡的影响。通过高通量Illumina RNA测序，筛选蟾毒灵作用人肝癌细胞后基因表达的变化，并进行生物信息学分析。结果：中药单体蟾毒灵可抑制人肝癌细胞增殖并诱导其凋亡;中药单体蟾毒灵可诱导不同基因的表达;基因本体（GO）富集分析表明，中药单体蟾毒灵可以影响细胞周期、凋亡等通路的变化;京都基因和基因组百科全书（KEGG）富集分析凋亡通路表明，钙离子诱导的细胞死亡途径中的钙蛋白酶表达明显上调。结论：中药单体蟾毒灵具有明显的抗肝癌细胞凋亡作用，并可通过作用相关信号通路中的多靶点参与此过程。

关键词 蟾毒灵;肝癌;增殖;凋亡;转录组学;体外;基因表达;生物信息学

Screening of Potential Targets of Bufalin against Hepatocellular Carcinoma Cells by Functional Genomics

LIN Jiamao1，ZHANG Chenyue2，BU Shuai3，LI Zhenxiang1，ZHAO Chenglong1，WANG Haiyong1

（1 Shandong Cancer Hospital and Institute，Ji′nan 250117，China; 2 Fudan University Shanghai Cancer Center，Shanghai 200032，China; 3 The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine，Ji′nan 250001，China）

Abstract Objective：This study aims to screen molecular targets of bufalin in promoting apoptosis of human hepatocellular carcinoma cells.Methods：The effect of bufalin on apoptosis and gene expression of human hepatocellular carcinoma PLC/PRF/5 cells was investigated.To be specific，the PLC/PRF/5 cells were treated with different concentration of bufalin for 24 h，48 h，and 72 h，respectively，and the proliferation and apoptosis of the cells were detected.Illumina high-throughput sequencing of RNA was performed to observe the changes of gene expression in the cells treated with bufalin，and the bioinformation of related genes was analyzed.Results：Bufalin inhibited the proliferation and induced the apoptosis of PLC/PRF/5 cells.It induced the expression of different genes.Gene Ontology（GO） term analysis showed that bufalin influenced cell cycle，apoptosis，and other pathways.Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway analysis suggested that calpain was significantly up-regulated in calcium-induced cell death pathway.Conclusion：Bufalin remarkably promotes the apoptosis of human hepatocellular carcinoma cells through multiple targets in related pathways.

Keywords Bufalin; Hepatocellular carcinoma; Proliferation; Apoptosis; Transcriptome; In vitro; Gene expression; Bioinformatics

中图分类号：R273;R285.5文献标识码：Adoi：10.3969/j.issn.1673-7202.2022.08.002

目前普遍认为肿瘤的发生发展是多因素所致的病理过程[1-2]。基因改变是肿瘤发生发展的重要原因[3-4]。目前临床中所用的许多化疗药物就是针对基因突变的分子而发挥良好的抗肿瘤功效[5-6]。

蟾毒灵是从中药蟾酥中提取的最具活性的抗肿瘤成分，已被证实对多种肿瘤具有很强的抗肿瘤作用[7-9]。研究表明，蟾毒灵可以抑制腫瘤细胞的增殖和迁移，诱导细胞凋亡和逆转耐药性[10-14]。……