纳米孔长读测序鉴定肝癌细胞中低氧特异性转录本  

Identification of Hypoxia-specific Transcripts in Liver Cancer Cells via Nanopore Long-read Sequencing

作　　者：高力 沈晟齐 高平 GAO Li;SHEN Shengqi;GAO Ping(School of Medicine,South China University of Technology,Guangzhou 510006,China;Medical Research Insti-tute,Guangdong Provincial People’s Hospital//Guangdong Academy of Medical Sciences//Southern Medical University,Guangzhou 510006,China)

机构地区：[1]华南理工大学医学院,广东广州510006 [2]广东省人民医院医学研究部//广东省医学科学院//南方医科大学,广东广州510006

出　　处：《中山大学学报(医学科学版)》2025年第1期88-97,共10页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(82103186)。

摘　　要：【目的】应用纳米孔长读长测序技术,深入分析正常肝实质细胞和肝癌细胞在低氧环境下的特异性转录组变化,以期阐明肿瘤细胞在低氧条件下生存与转移的内在机制,从而为肿瘤的精准靶向治疗开辟新的视角与策略。【方法】实验选取正常肝实质细胞系THLE-3与肝癌细胞系Hep3B作为研究对象,分别置于常氧与低氧条件下培养48 h后,提取其总RNA。随后,采用纳米孔测序技术,对这两种细胞系在不同氧浓度下的转录组进行高通量、高精度的测序分析。【结果】本研究成功构建了基于纳米孔三代测序技术的低氧转录本数据集,展现了前所未有的测序精度。通过GO(gene ontology)富集分析,我们系统地挖掘了参与低氧响应的关键生物通路(P<0.05)。进一步地,结合分子动力学模拟方法,我们深入探究了溶质载体家族1成员5(SLC1A5)在低氧特异性转录本翻译过程中,其蛋白质结构所发生的动态变化,为理解其功能调控提供了直接证据。【结论】纳米孔长读长测序技术的应用,不仅成功捕捉到了低氧条件下mRNA的特征性表达模式及特异性转录本,同时为深入探索低氧微环境下的转录组复杂性提供了强有力的技术支撑。在进一步结合蛋白结构模拟以及分子动力学的基础上,对低氧微环境中的蛋白结构提出新的探索路径。本研究的发现,不仅丰富了低氧特异性转录组学的研究内容,为低氧特异性蛋白结构探索提供了更可靠的数据基础,更为未来肿瘤的靶向治疗策略提供了低氧特异性的潜在靶点。【Objective】Leveraging the advanced capabilities of nanopore long-read sequencing technology,our study undertook a comprehensive analysis of the distinct transcriptomic alterations occurring in normal liver parenchymal cells and liver cancer cells subjected to hypoxic conditions.The primary goal was to elucidate the underlying mechanisms governing tumor cell survival and metastasis in low-oxygen environments,thereby paving the way for innovative targeted cancer therapies.【Methods】The normal liver parenchymal cell line THLE-3 and the hepatocellular carcinoma cell line Hep3B were chosen as the focal points of this investigation.Following a 48-hour incubation period in both normoxic and hypoxic conditions,total RNA was extracted from these cells.Subsequently,we employed nanopore sequencing technology to conduct a high-throughput,high-fidelity analysis of the transcriptomes of these two cell lines across different oxygen levels.【Results】This study established a hypoxic transcriptome dataset using third-generation nanopore sequencing technology,achieving an unprecedented level of sequencing accuracy.By conducting a Gene Ontology(GO)enrichment analysis,we systematically identified and explored the key biological pathways associated with the hypoxic response(P<0.05).Furthermore,we integrated molecular dynamics simulation techniques to gain deeper insights into the dynamic structural changes of Solute Carrier Family 1 Member 5(SLC 1 A 5)during the translation of hypoxic-specific subtypes,providing direct evidence to elucidate its functional regulation.【Conclusion】The application of nanopore long-read sequencing technology has proven to be a powerful tool,not only successfully capturing the distinctive expression patterns and specific subtypes of mRNA under hypoxic conditions,but also offering robust technical support for delving into the intricate transcriptomic landscape of hypoxic microenvironments.By further integrating protein structure simulations and molecular dynamics,we have proposed novel avenues

关 键 词：纳米孔测序 低氧 转录本 可变剪切 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]