Transcriptome profile and its partly verification of human hepatocellular carcinoma cells exposed to Yuzhizi () seed
Fan Xiaoyan1, L I Mingzhe1, Chen Long2
1Department of Experimental Traditional Chinese Medicine, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Objective:
To investigate the efficacy of Yuzhizi seed extract (FAQSE) on inhibiting the proliferation of hepatocellular carcinoma (HCC) cells in vitro and to explore the anti-HCC action mechanism of FAQSE.
Methods:
Human HCC HepG2 and Huh7 cells were used to investigate the anti-HCC effect of FAQSE. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method was used to measure cell viability. Affymetrix microarray was adopted to detect the expression of transcriptome. The differentially expressed genes (DEGs) of each cell line were identified. For co-DEGs of both cell lines, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were enriched using the Database for Annotation, Visualization and Integrated Discovery (DAVID), and the network analysis of protein-protein interaction (PPI) was mapped using the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape software. Some important genes in the PPI network of co-DEGs were selected to verify by quantitative real-time reverse transcription-polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay.
Results:
FAQSE decreased the viability of HepG2 and Huh7 cells. There were 211 co-upregulated and 86 co-downregualted genes in both cell lines after FAQSE treatment. The enriched GO terms of co-upregulated DEGs were primarily involved cell-cell adhesion, viral process, transcription initiation from RNA polymerase II promoter, positive regulation of transcription from RNA polymerase II promoter and actin cytoskeleton organization. The GO terms of co-downregulated DEGs were mainly enriched in the processes of SRP-dependent cotranslational protein targeting to membrane, viral transcription, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, translational initiation and rRNA processing. Main KEGG pathways of co-upregulated DEGs were endocytosis, glutathione metabolism, protein processing in endoplasmic reticulum, synaptic vesicle cycle and lysosome. The major KEGG pathways of co-downregulated DEGs were ribosome, biosynthesis of amino acids, arginine and proline metabolism, systemic lupus erythematosus and complement and coagulation cascades. The top 10 co-DEGs with high hub nodes in STRING analysis were ribosomal protein S27a, transferrin, ribosomal protein S20, ribosomal protein L9, protein phosphatase 2 regulatory subunit B alpha, transthyretin, thioredoxin reductase 1, ribosomal protein L3, ribophorin I and ribosomal protein L24. Alpha-fetoprotein (AFP) was also co-downregulated and contained in the PPI network. The mRNA and protein expression of most verified genes was consistent with the results of co-DEGs analysis. And the AFP level was significantly reduced after FAQSE treatment.
Conclusions:
A series of genes and pathways of HepG2 and Huh7 cells were changed after FAQSE treatment, which might be the targets of FAQSE against HCC and worthy of further study. AFP might be important one of them.
