Triterpenes from Ganoderma lucidum inhibit hepatocellular carcinoma by regulating enhancer-associated lncRNA in vivo.

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE 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. AIM OF THE STUDY 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.