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目的 ：基于前期单细胞RNA测序揭示的肝细胞癌高度异质性，从恶性上皮细胞中筛选关键差异表达基因并进行验证，为确认作为潜在生物标志物或治疗靶点提供依据。方法：对前期已构建的肝细胞癌单细胞转录组数据进行分析，聚焦于恶性上皮细胞群，通过生物信息学方法筛选显著差异表达基因。在肝癌细胞株中，利用实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction,qRT-PCR）技术对候选差异基因进行体外表达水平验证。结果：单细胞数据分析鉴定出神经降压素（Neurotensin,NTS）、纤维蛋白原β链（fibrinogen beta chain,FGB）、长链酰基辅酶A合成酶4(long-chain acyl-CoA synthetase 4,ACSL4)、母系表达基因3(maternally expressed gene 3,MEG3)、瘦素受体（leptin receptor,LEPR）和Janus激酶3(Janus kinase 3,JAK3)为肝癌恶性上皮细胞中特异性高表达的关键基因。qRT-PCR实验结果与测序数据高度一致，证实这些基因在肝癌细胞株中的异常表达模式，从而锁定数个在转录组和转录后水平均稳定异常的潜在靶点。结论：通过整合单细胞测序分析与体外分子实验验证，构建从生物信息学发现到实验验证的完整研究闭环。所验证的关键差异基因为深入理解肝癌的发病机制及其作为潜在的诊断标志物或治疗靶点提供了有价值的依据。
Abstract：Objective: Based on previous single-cell RNA sequencing(scRNA-seq) data revealing the high heterogeneity of hepatocellular carcinoma(HCC), this study aimed to identify and validate key differentially expressed genes(DEGs) in malignant epithelial cells, in order to explore their potential as biomarkers or therapeutic targets. Methods: Using previously established single-cell transcriptomic data of HCC, we conducted in-depth analyses focusing on malignant epithelial cell populations and screened for significantly differentially expressed genes through bioinformatics approaches.The expression levels of selected candidate genes were subsequently validated in hepatocellular carcinoma cell lines using quantitative real-time polymerase chain reaction(qRT-PCR). Results: The single-cell data analysis successfully identified Neurotensin(NTS), fibrinogen beta chain(FGB), long-chain acyl-CoA synthetase 4(ACSL4), maternally expressed gene 3(MEG3), leptin receptor(LEPR), and Janus kinase 3(JAK3) as key genes with specifically high expression in malignant epithelial cells of liver cancer. qRT-PCR validation results were highly consistent with the sequencing data, confirming the aberrant expression patterns of these genes in HCC cell lines and pinpointing a subset of genes exhibiting stable dysregulation at both transcriptional and post-transcriptional levels. Conclusion: By integrating single-cell transcriptomic analysis with in vitro molecular validation, this study established a complete research loop from bioinformatic discovery to experimental confirmation. The validated DEGs provide a solid molecular foundation for understanding the pathogenesis of HCC and offer valuable evidence supporting their potential as diagnostic biomarkers or therapeutic targets.
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基本信息:
DOI：10.19767/j.cnki.32-1412.2025.06.004
中图分类号:R735.7
引用信息:
[1]李鸣.肝细胞癌上皮细胞特征基因鉴定及其分子机制研究[J].交通医学,2025,39(06):567-572.DOI:10.19767/j.cnki.32-1412.2025.06.004.
基金信息:
南通市科技计划项目（MS2023081）; 江苏省卫生委员会科学研究项目（ZDB2020003）