Central Laboratory, Shanghai Skin Disease Hospital, School of Medicine, Tongji University, Shanghai, 200443, China; Institute of Basic Medical Research, Naval Medical University, Shanghai, 200433, China.
Central Laboratory, Shanghai Skin Disease Hospital, School of Medicine, Tongji University, Shanghai, 200443, China.
J Ethnopharmacol. 2025 Jan 10;336:118706. doi: 10.1016/j.jep.2024.118706. Epub 2024 Aug 24.
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.
灵芝（Ganoderma lucidum）已被广泛用作多种肿瘤的抗肿瘤治疗的辅助药物。灵芝的生物活性成分主要包括三萜类化合物，如灵芝酸 A、灵芝酸 B、灵芝烯酸 A、灵芝烯酸 B、灵芝烯酸 D 和灵芝酸 X。然而，灵芝在肝细胞癌（HCC）治疗中的作用和潜在机制往往具有挑战性。
通过体内和体外实验，探讨增强子相关长非编码 RNA（en-lncRNA）在灵芝治疗 HCC 中的潜在作用和机制。
建立荷 Hepa1-6 的 C57BL/6 小鼠模型，评估灵芝治疗 HCC 的疗效。Ki67 和 TUNEL 染色用于检测体内肿瘤细胞的增殖和凋亡。实施小鼠 lncRNA 4*180K 阵列，以鉴定灵芝处理肿瘤小鼠中差异表达（DE）的 lncRNA 和 mRNA。构建 lncRNA-mRNA 共表达网络和生物信息学分析，以选择核心 en-lncRNA 及其相邻基因。UPLC-MS 方法用于鉴定灵芝的三萜类化合物，体外实验用于验证哪种三萜类单体调节肿瘤细胞中的 en-lncRNA。最后，使用稳定的敲低/过表达细胞系来验证 en-lncRNA 与邻近基因之间的关系。
Ki67 和 TUNEL 染色表明灵芝显著抑制肿瘤生长，抑制体内肿瘤细胞增殖并诱导细胞凋亡。转录组分析显示灵芝处理的肿瘤小鼠中存在 126 个与 454 个共表达 mRNA 高相关的 DE lncRNA。基于 lncRNA-mRNA 网络和 qRT-PCR 验证，选择了 6 个核心 lncRNA，并认为其与灵芝治疗高度相关。生物信息学分析表明 FR036820 和 FR121302 可能作为增强子发挥作用，qRT-PCR 结果表明 FR121302 可能增强 HCC 中的 Popdc2 mRNA 水平。此外，通过 UPLC-MS 方法鉴定了灵芝的 6 种主要三萜类单体，体外实验表明 Ganoderenic 酸 A 和 Ganoderenic 酸 B 分别显著抑制 FR121302 和 Popdc2。敲低/过表达结果表明 FR121302 激活并增强 Popdc2 的表达水平，而 Ganoderenic 酸 A 和 Ganoderenic 酸 B 则显著抑制 FR121302 并降低 Hepa1-6 细胞中的 Popdc2 水平。
增强子相关的 lncRNA 在肝癌发生过程中作为增强子发挥关键作用，灵芝的三萜类化合物通过调节 en-lncRNA 显著抑制肿瘤细胞增殖并诱导细胞凋亡。我们的研究表明，Ganoderenic 酸 A 和 Ganoderenic 酸 B 抑制 en-lncRNA FR121302 的表达可能是灵芝抑制肝细胞癌生长的关键策略之一。
