| objective: To investigate the effect of natural compound N-transferuloyloctopamine(FO)on the proliferation,migration and invasion of hepatocellular carcinoma cell lines Huh7 and HCCLM3,and to explore the role of FO in inhibiting the growth of liver cancer cells and its molecular mechanisms.Methods: Hepatocellular carcinoma cells Huh7 and HCCLM3 were cultured in vitro,and CCK-8 assays were used to verify the inhibitory effect of different concentrations of FO on the proliferation of HCC cells and the IC50 was calculated;The effects of FO on the migration and invasion ability of hepatocellular carcinoma cells were observed using wound-healing and Transwell assays;Western Blot was used to verify the phosphorylation levels of AKT,p38 MAPK,ERK1/2,and p65 in HCC and the expression levels of key proteins Slug,Snail,Ncadherin,and E-cadherin in the EMT pathway after FO was treated;Flow cytometry was performed to detect the role of FO on apoptosis,and using RNAseq technology to screen differentially expressed genes in Huh7 cells after FO treated;GO and KEGG enrichment analysis of differentially expressed genes using DAVID bioinformatics tools to further clarify the functions and signaling pathways of differential gene regulation,combined with GO and KEGG analysis data,to screen potential target genes for FO regulation.Results: 1.Different concentrations of FO(0mM,0.1mM,0.5mM,1mM,2mM,4mM,10 mM,20mM,50mM)can significantly inhibit Huh7 and HCCLM3 cells,this effect is dose-dependent(p<0.05).The IC50 of Huh7 and HCCLM3 cells were determined to be 2.00 mM and 2.27 mM,respectively.2.Transwell assay showed that FO could significantly reduce the invasion of hepatocellular carcinoma cells.When Huh7 and HCCLM3 cells were treated with FO(2.00 mM,2.27mM)for 48 h,the number of cells crossing the matrix membrane was significantly less than NC group(p<0.05).However,wound-healing assay showed that FO can delay the metastasis of liver cancer cells,but this inhibitory effect is not statistically significant.3.Western Blot data showed that phosphorylation of AKT and p38 MAPK was inhibited.Moreover,in the EMT signal pathway,the expression of E-cadherin was up-regulated and Slug was decreased,but the expression of Snail and Ncadherin did not change.When hepatocellular carcinoma cells were treated with FO for 24 hours(p< 0.05).4.Flow cytometry showed that FO could promote the apoptosis of HCC cells,and this effect was time-dependent.Compared with the treatment with FO for 24 hours,the apoptosis rate was significantly increased in 48 hours.5.RNA-seq data showed that there were 317 differentially expressed genes in Huh7 cells after FO was treated,of which 188 genes were up-regulated and 129 genes were down-regulated.GO and KEGG enrichment analysis found that the main GO terms enriched by these DEGs include: cell proliferation,apoptosis,cell cycle,etc.The main enriched KEGG pathways include: PI3K-AKT,MAPK,and apoptosis.Combined the data of GO and KEGG enrichment analysis,CDKN1 A,BBC3,DDIT3,and NOXA were screened as potential hub genes of FO.Conclusion: In HCC,FO can inhibit proliferation,invasion,migration and promote apoptosis.FO inhibits the proliferation of liver cancer cells by regulating the phosphorylation levels of AKT and p38 MAPK,regulates the expression of Ecadherin and Slug to inhibit the invasion of liver cancer cells by mediating the EMT signaling pathway.In addition,transcriptome sequencing results revealed that a group of important key genes and signaling pathways changed after FO intervention.Through our comprehensive bioinformatics analysis,we found that FO can mediate the PI3K-AKT and apoptosis signal pathway of liver cancer cells by regulating the expression of CDKN1 A,BBC3,DDIT3,and NOXA.These candidate genes are expected to be potential targets for the diagnosis and treatment of liver cancer.This provides theoretical basis and experimental data for the research of targeted treatment of liver cancer and natural antitumor drugs. |
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