Transcriptome analysis of ethanol extract of Akebia trifoliate(Thunb.)Koidz on cell proliferation inhibitory functions and apoptosis promotion in human liver cells
Objective To investigate the effects of ethanol extract of Akebia trifoliate(Thunb.)Koidz(EEATK)on the proliferation and apoptosis of human hepatocellular carcinoma Hep3B and Huh-7 cells and to explore its underlying mechanism.Methods Human Hep3B cells and Huh-7 cells were cultured in vitro and separated into control group,Sorafenib group(5 μmol/L),and EEATK groups(0.10 mg/mL,0.15 mg/mL,0.20 mg/mL,0.3 mg/mL)and given the corresponding drug interventions.A CCK-8 assay was used to measure the impact of the different interventions on the proliferation of Hep3B cells and Huh-7 cells to screen the optimal action-inducing concentrations for subsequent experiments.EdU staining assay and colony formation assay were used to explore the effect of EEATK on proliferation,and Annexin V-FITC/PI double-staining assay was applied for apoptotic rate analysis.Transcriptome sequencing(RNA-seq)technology was used to analyze differentially expressed genes(DEGs)related to cell proliferation and apoptosis in the control group and EEATK(0.15 mg/mL)groups of Hep3B cells.DEGs were analyzed for function with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.The Comparative Toxicogenomics Database(CTD)was used to validate the expression of key proteins related to cell proliferation and apoptosis,and the findings were verified by qRT-PCR.Results Compared with the control group,different concentration of EEATK significantly inhibited the activity of Hep3B and Huh-7 cells(P＜0.01).Hep3B cells were treated with 0.15 mg/mL EEATK,the EdU-positive cell rate and clone formation rate significantly decreased(all P＜0.01).At the same time,the apoptotic rate of the EEATK group significantly increased(P＜0.01).Transcriptome sequencing of Hep3B cells showed that EEATK induced significant changes in the expression of 1577 genes(P＜0.01),of which 942 were up-regulated and 635 were down-regulated compared with the control group.GO functional enrichment analysis revealed that the DEGs were mainly enriched for cholesterol synthesis,inflammation,and extracellular matrix.KEGG pathway analysis showed that EEATK plays an anti-tumor role,mainly through the TFG-β and NF-κB signaling pathways.CTD and qRT-PCR analysis showed that EEATK significantly down-regulated the expression of apoptosis-related genes such as BIRC5 and CDK1(P＜0.01)and significantly upregulated the expression of CDKN1A and EGLN3(P＜0.01).At the same time,EEATK caused the significant downregulation of cell-proliferation-related genes such as FAM83D and MKI-67(P＜0.01),and significantly upregulated the expression of MYC and FOXC1(P＜0.01).Conclusions EEATK can inhibit cell proliferation and induce apoptosis in a manner that may be related to the regulation of TGF-β and NF-κB signal pathway-related gene expression.
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