Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Science, Northeast Forestry University, Harbin 150040, China.
Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Science, Northeast Forestry University, Harbin 150040, China; State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China; Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Harbin 150040, China.
Biochim Biophys Acta Gen Subj. 2021 Jul;1865(7):129911. doi: 10.1016/j.bbagen.2021.129911. Epub 2021 Apr 20.
Ellagic acid (EA) possesses prominent inhibitory activities against various cancers, including hepatocellular carcinoma (HCC). Our recent study demonstrated EA's activities in reducing HCC cell proliferation and tumor formation. However, the mechanisms of EA to exert its anticancer activities and its primary targets in cancer cells have not been systematically explored.
Cell proliferation assay and flow cytometric analysis were used to examine the effects of EA treatment on viability and apoptosis, respectively, of HepG2 cells. RNA-seq studies and associated pathway analyses by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to determine EA's primary targets. Differentially expressed genes (DEG) in EA-treated HepG2 cells were verified by RT-qPCR and Western blot. Integrative analyses of the RNA-seq dataset with a TCGA dataset derived from HCC patients were conducted to verify EA-targeted genes and signaling pathways. Interaction network analysis of the DEGs, shRNA-mediated knockdown, cell viability assay, and colony formation assay were used to validate EA's primary targets.
EA reduced cell viability, caused DNA damage, and induced cell cycle arrest at G1 phase of HepG2 cells. We identified 5765 DEGs encoding proteins with over 2.0-fold changes in EA-treated HepG2 cells by DESeq2. These DEGs showed significant enrichment in the pathways regulating DNA replication and cell cycle progression. As primary targets, p21 was significantly upregulated, while MCM2-7 were uniformly downregulated in response to EA treatment. Consistently, p21 knockdown desensitized liver cells to EA in cell viability and colony formation assays.
EA induced G1 phase arrest and promoted apoptosis of HCC cells through activating the p21 gene and downregulating the MCM2-7 genes, respectively.
The discoveries in this study provide helpful insights into developing novel strategies in the therapeutic treatment of HCC patients.
鞣花酸（EA）对多种癌症具有显著的抑制活性，包括肝细胞癌（HCC）。我们最近的研究表明，EA 能够减少 HCC 细胞的增殖和肿瘤形成。然而，EA 发挥抗癌活性的机制及其在癌细胞中的主要靶点尚未得到系统探索。
使用细胞增殖测定和流式细胞术分析分别检测 EA 处理对 HepG2 细胞活力和凋亡的影响。RNA-seq 研究以及通过基因本体论（GO）和京都基因与基因组百科全书（KEGG）进行的相关途径分析，用于确定 EA 的主要靶点。通过 RT-qPCR 和 Western blot 验证 EA 处理的 HepG2 细胞中差异表达基因（DEG）。对来自 HCC 患者的 TCGA 数据集与 RNA-seq 数据集进行综合分析，以验证 EA 靶向基因和信号通路。使用 DEG 的互作网络分析、shRNA 介导的敲低、细胞活力测定和集落形成测定，验证 EA 的主要靶点。
EA 降低了 HepG2 细胞的活力，导致 DNA 损伤，并诱导细胞周期停滞在 G1 期。通过 DESeq2 我们在 EA 处理的 HepG2 细胞中鉴定到 5765 个编码蛋白的 DEGs，其变化倍数超过 2.0 倍。这些 DEGs 在调节 DNA 复制和细胞周期进程的途径中表现出显著的富集。作为主要靶点，p21 显著上调，而 MCM2-7 则在 EA 处理后均匀下调。一致地，p21 敲低使肝细胞对 EA 在细胞活力和集落形成测定中变得敏感。
EA 通过分别激活 p21 基因和下调 MCM2-7 基因，诱导 HCC 细胞 G1 期阻滞并促进细胞凋亡。
本研究的发现为开发 HCC 患者治疗的新策略提供了有价值的见解。
