Transcriptome Analysis Of Signaling Pathways Targeted By Ellagic Acid In Hepatocellular Carcinoma Cells

Ellagic Acid(EA)is an emerging natural small molecule medicine that inhibits the development of various cancers,including hepatocellular carcinoma(HCC).In recent years,accumulating researches demonstrated the mechanisms of EA to exert its functions and indicated its primary targets in cancer cells.Our recent study revealed that EA possesses the activities in suppressing HCC cell proliferation and tumor formation;EA can also induce cell apoptosis,DNA damage and cell cycle arrest of HCC cells,but has very low toxicity in normal liver cells.To explore the intracellular changes in EA-treated HCC cells at the transcriptional level,we carried out the RNA sequencing(RNA-seq)and differential gene expression analysis of EA-treated HepG2 cells,and compared the RNA-seq data of EA-treated HepG2 cells with the dataset of liver hepatocellular carcinoma from The Cancer Genome Atlas(TCGA-LIHC).1.Using the DESeq method,we discovered that EA-treated HepG2 cells had 6127 differentially expressed genes(DEGs),including 3385 up-regulated and 2742 down-regulated genes,compared to the DMSO-treated cells.Meanwhile,we also found that 2179 DEGs in tumor tissues of the TCGA-LIHC dataset,including 1306 up-regulated and 873 down-regulated genes,compared to the para-carcinoma tissues.2.EA showed relatively low toxicity to normal liver cells and relatively high toxicity to HCC cells.To explore EA-targeted DEGs in HCC cells,we overlapped the DEGs of the RNA-seq dataset of EA-treated HepG2 cells with the DEGs of the TCGA-LIHC dataset.Among the upregulated or downregulated genes of the RNA-seq dataset of EA-treated cells,we identified 193 reduced and 404 increased genes with reverse changes,respectively,in the TCGA-LIHC dataset.Then,we used the GO and KEGG enrichment analyses to analyze the data of these reversely changed genes and found that the most enriched terms fell in the genes regulating cell cycle and DNA replication.3.We selected 24 reversely changed DEGs involved cell cycle and DNA replication.The expression changes of most of the selected genes could be confirmed by reverse transcription and quantitative polymerase chain reaction(RT-qPCR),except INHBA,which could not be detected by RT-qPCR.These 23 genes that could be confirmed by RT-qPCR are thus considered as the target genes of EA to cause cell growth inhibition.We used the survival analysis and Cox's multivariate regression analysis to process these genes based on the TCGA-LIHC dataset.We discovered that the high-risk expression of all 23 genes could significantly reduce 20.5%5-year overall survival rates of the patients and increased risk of death in the Cox's multivariate regression analysis.In addition,17 of these genes significantly correlated with clinical outcomes of the liver cancer patients in the survival analysis.These results provided theoretical supports to verifying the potential of EA as a therapeutic and understanding its primary targets in clinical treatment of HCC.In summary,we determined the primary targets of EA as the genes regulating the DNA synthesis and cell cycle progression through integrative analyses of the datasets of EA-treated HepG2 cells and TCGA-LIHC.Based on the survival analysis and Cox's proportional hazards regression model analysis,we propose that EA,as a potential clinical therapeutic,may target the genes in these two biological processes to exert its inhibitory activity against HCC.