灵芝（G. lucidum）已广泛用作多种肿瘤抗肿瘤治疗的辅助药物。灵芝的生物活性成分主要为三萜类化合物，如灵芝酸A、灵芝酸B、灵芝酸A、灵芝酸B、灵芝酸D、灵芝酸X等。但灵芝的功效及作用机制尚不清楚。在肝细胞癌（HCC）治疗中经常面临挑战。通过体内和体外实验探讨增强子相关lncRNA（en-lncRNA）在灵芝治疗HCC中的潜在作用和机制。Hepa1-6-bearing C57 BL/建立6只小鼠模型来评价灵芝治疗HCC的疗效。 Ki67和TUNEL染色检测体内肿瘤细胞增殖和凋亡。使用小鼠lncRNA 4*180K阵列来鉴定灵芝治疗的肿瘤小鼠的差异表达（DE）lncRNA和mRNA。利用构建的lncRNA-mRNA共表达网络和生物信息学分析来选择核心en-lncRNA及其邻近基因。采用UPLC-MS方法对灵芝三萜进行鉴定，并通过体外实验验证哪些三萜单体在肿瘤细胞中调控en-lncRNA。最后，使用稳定的敲低/过表达细胞系来证实en-lncRNA与邻近基因之间的关系。Ki67和TUNEL染色表明灵芝在体内显着抑制肿瘤生长，抑制细胞增殖并诱导细胞凋亡。转录组分析显示，在灵芝治疗的肿瘤小鼠中，存在 126 个 DE lncRNA 与 454 个共表达 mRNA 高度相关。基于lncRNA-mRNA网络和qRT-PCR验证，选择了6个核心lncRNA，并认为与灵芝处理高度相关。生物信息学分析显示 FR036820 和 FR121302 可能充当增强子，qRT-PCR 结果表明 FR121302 可能增强 HCC 中 Popdc2 mRNA 水平。此外，通过UPLC-MS方法鉴定了灵芝的6个主要三萜单体，体外实验表明灵芝酸A和灵芝酸B分别显着抑制FR121302和Popdc2。敲除/过表达结果表明，FR121302激活并增强Popdc2表达水平，灵芝酸A和灵芝酸B显着抑制FR121302并降低Hepa1-6细胞中Popdc2水平。增强子相关lncRNA在肝癌发生过程中作为增强子发挥着至关重要的作用。灵芝中的三萜类化合物通过调节en-lncRNA显着抑制肿瘤细胞增殖并诱导细胞凋亡。我们的研究表明，灵芝酸A和灵芝酸B抑制en-lncRNA FR121302可能是灵芝抑制肝细胞癌生长的关键策略之一。版权所有©2024。由Elsevier B.V.出版。

Ganoderma lucidum (G. lucidum) has been widely used as adjuvant of anti-tumor therapy for variety tumors. The bioactive ingredients of G. lucidum mainly include triterpenes, such as Ganoderic acid A, Ganoderic acid B, Ganoderenic acid A, Ganoderenic acid B, Ganoderenic acid D, and Ganoderic acid X. However, the effects and underlying mechanisms of G. lucidum are often challenging in hepatocellular carcinoma (HCC) treatment.To explore the potential role and mechanism of enhancer-associated lncRNAs (en-lncRNAs) in G. lucidum treated HCC through the in vivo and in vitro experiments.Hepa1-6-bearing C57 BL/6 mice model were established to evaluate the therapeutic efficacy of G. lucidum treated HCC. Ki67 and TUNEL staining were used to detect the tumor cell proliferation and apoptosis in vivo. The Mouse lncRNA 4*180K array was implemented to identify the differentially expressed (DE) lncRNAs and mRNAs of G. lucidum treated tumor mice. The constructed lncRNA-mRNA co-expression network and bioinformatics analysis were used to selected core en-lncRNAs and its neighboring genes. The UPLC-MS method was used to identify the triterpenes of G. lucidum, and the in vitro experiments were used to verify which triterpene monomers regulated en-lncRNAs in tumor cells. Finally, a stable knockdown/overexpression cell lines were used to confirm the relationship between en-lncRNA and neighboring gene.Ki67 and TUNEL staining demonstrated G. lucidum significantly inhibited tumor growth, suppressed cell proliferation and induced apoptosis in vivo. Transcriptomic analysis revealed the existence of 126 DE lncRNAs high correlated with 454 co-expressed mRNAs in G. lucidum treated tumor mice. Based on lncRNA-mRNA network and qRT-PCR validation, 6 core lncRNAs were selected and considered high correlated with G. lucidum treatment. Bioinformatics analysis revealed FR036820 and FR121302 might act as enhancers, and qRT-PCR results suggested FR121302 might enhance Popdc2 mRNA level in HCC. Furthermore, 6 main triterpene monomers of G. lucidum were identified by UPLC-MS method, and in vitro experiments showed FR121302 and Popdc2 were significantly suppressed by Ganoderenic acid A and Ganoderenic acid B, respectively. The knock/overexpression results demonstrated that FR121302 activating and enhancing Popdc2 expression levels, and Ganoderenic acid A and Ganoderenic acid B dramatically suppressed FR121302 and decreased Popdc2 level in Hepa1-6 cells.Enhancer-associated lncRNA plays a crucial role as an enhancer during hepatocarcinogenesis, and triterpenes of G. lucidum significantly inhibited tumor cell proliferation and induced apoptosis by regulating en-lncRNAs. Our study demonstrated Ganoderenic acid A and Ganoderenic acid B suppressed en-lncRNA FR121302 may be one of the critical strategies of G. lucidum inhibit hepatocellular carcinoma growth.Copyright © 2024. Published by Elsevier B.V.