Transcriptome analysis of signaling pathways targeted by Ellagic acid in hepatocellular carcinoma cells.

Abstract

BACKGROUND\nEllagic 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.\n\n\nMETHODS\nCell 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.\n\n\nRESULTS\nEA 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.\n\n\nCONCLUSION\nEA induced G1 phase arrest and promoted apoptosis of HCC cells through activating the p21 gene and downregulating the MCM2-7 genes, respectively.\n\n\nGENERAL SIGNIFICANCE\nThe discoveries in this study provides helpful insights into developing novel strategies in the therapeutic treatment of HCC patients.