Amentoflavone reverses epithelial-mesenchymal transition in hepatocellular carcinoma cells by targeting p53 signalling pathway axis

Abstract

Epithelial-mesenchymal transition (EMT) and its reversal process are important potential mechanisms in the development of HCC. Selaginella doederleinii Hieron is widely used in Traditional Chinese Medicine for the treatment of various tumours and Amentoflavone is its main active ingredient. This study investigates the mechanism of action of Amentoflavone on EMT in hepatocellular carcinoma from the perspective of bioinformatics and network pharmacology. Bioinformatics was used to screen Amentoflavone-regulated EMT genes that are closely related to the prognosis of HCC, and a molecular prediction model was established to assess the prognosis of HCC. The network pharmacology was used to predict the pathway axis regulated by Amentoflavone. Molecular docking of Amentoflavone with corresponding targets was performed. Detection and evaluation of the effects of Amentoflavone on cell proliferation, migration, invasion and apoptosis by CCK-8 kit, wound healing assay, Transwell assay and annexin V-FITC/propidium iodide staining. Eventually three core genes were screened, inculding NR1I2, CDK1 and CHEK1. A total of 590 GO enrichment entries were obtained, and five enrichment results were obtained by KEGG pathway analysis. Genes were mainly enriched in the p53 signalling pathway. The outcomes derived from both the wound healing assay and Transwell assay demonstrated significant inhibition of migration and invasion in HCC cells upon exposure to different concentrations of Amentoflavone. The results of Annexin V-FITC/PI staining assay showed that different concentrations of Amentoflavone induces apoptosis in HCC cells. This study revealed that the mechanism of Amentoflavone reverses EMT in hepatocellular carcinoma, possibly by inhibiting the expression of core genes and blocking the p53 signalling pathway axis to inhibit the migration and invasion of HCC cells.

Keywords: Amentoflavone; bioinformatics; epithelial‐mesenchymal transition; hepatocellular carcinoma; network pharmacology; p53 signalling pathway axis.

FIGURE 1

The schematic diagram of the research methodology and workflow of the present study of Amentoflavone acting on EMT in hepatocellular carcinoma.

FIGURE 2

The screening of key genes, construction and evaluation of prognostic models. (A) The volcano map of hepatocellular carcinoma differential genes; (B) the Venn diagram of intersecting genes; (C) unpaired sample t‐test results for intersecting genes; (D) paired‐sample t‐test results for intersecting genes. (E) The visualization results of Lasso regression for intersecting genes; (F) time‐dependent receiver operating characteristic (ROC) curve of the Risk scoring system for core genes; (G) Kaplan–Meier survival of the Risk scoring system for core genes; (H) forest plots for single and multifactorial Cox regression analysis of core genes; (I) nomogram of the predictive model; (J) calibration analysis of nomogram prediction.

FIGURE 3

Network Pharmacology analysis results of Amentoflavone on Hepatocellular Carcinoma through modulation of EMT. (A) PPI network; (B) GO enrichment analysis (BP, biological process; CC, cell components; MF, molecular function); (C) KEGG enrichment analysis; (D) Gene enrichment map of the p53 signalling pathway (CHK1 also known as CHEK1, Cdc2 also known as CDK1, IGF is IGF1).

FIGURE 4

Docking diagram of Amentoflavone with core targets. (A) 2D chemical structure of Aamentoflavone; (B) Docking diagram of Amentoflavone and CHEK1; (C) Docking diagram of Amentoflavone and CDK1; (D) Docking diagram of Amentoflavone and IGF1.

FIGURE 5

Establishment and verification of HCC EMT model cells. (A) Representative images of H&E staining of HCC EMT model cells. (B) Statistical results on the mRNA expression levels of EMT‐regulated genes. (C, D) Protein electropherogram and statistical results on the protein expression levels of EMT‐regulated genes. All data are presented as mean ± standard deviation (SD; n = 3). (*p < 0.05 when compared to the Control group; **p < 0.01 when compared to the Control group).

FIGURE 6

Cytotoxicity of Amentoflavone in HCC cells. (A) Cytotoxicity assay results of Amentoflavone on MHCC97H cell; (B) Cytotoxicity assay results of Amentoflavone on MHCCLM3 cell. All data are presented as mean ± standard deviation (SD; n = 4).

FIGURE 7

Amentoflavone reverses the EMT in HCC cells. (A–D) Representative images and statistical results of wound healing assay; (E, F) Representative images and statistical results of Transwell assay. All data are presented as mean ± standard deviation (SD; n = 3). (*p < 0.05, **p < 0.01, and n s., not significant).

FIGURE 8

Amentoflavone induces apoptosis in HCC cells. (A) The detection of apoptosis of MHCC97H cells by Annexin V‐FITC/PI staining and flow cytometry; (B) Statistical data chart of apoptosis rate of MHCC97H cells; (C) Detection of apoptosis of MHCCLM3 cells by Annexin V‐FITC/PI staining and flow cytometry; (D) Statistical data chart of apoptosis rate of MHCCLM3 cells. All data are presented as mean ± standard deviation (SD; n = 3). (*p < 0.05, **p < 0.01, and n s., not significant).

FIGURE 9

Amentoflavone‐reversed EMT in HCC cells is mediated by the p53 signalling pathway axis. (A, B) Protein electropherogram of EMT‐regulated genes; (C) Statistical results of protein express levels; (D, E) Protein electropherogram of p53 signalling pathway proteins; (F) Statistical results of protein express levels. All data are presented as mean ± standard deviation (SD; n = 3). (*p < 0.05 when compared to the Control group; **p < 0.01 when compared to the Control group).

FIGURE 10

Anti‐epithelial‐mesenchymal transition mechanism of Amentoflavone in TGF‐β1 treated HCC cells.