Arecoline promotes proliferation and migration of human HepG2 cells through activation of the PI3K/AKT/mTOR pathway

Abstract

Background: Arecoline is a well-known risk factor for oral submucosal fibrosis and cancer. However, the mechanistic correlation between arecoline and hepatocellular cancer remains elusive. Here, we investigated the effect of arecoline on the proliferation and migration of human HepG2 hepatoma cells and its potential oncogenic mechanisms.

Methods: Bioinformatic technologies were used to identify the deferentially expressed miRNAs (DE-miRNAs) and hub target genes of arecoline-induced cancers. These DE-miRNAs, hub genes and pathway were proved in arecoline-treated HepG2 cells.

Results: A total of 86 DE-miRNAs and 460 target genes were identified. These target genes are associated with DNA-templated regulation of transcription and other biological processes. Significant molecular functions were protein binding, calcium ion binding, and enrichment in the nucleus and cytoplasm. These genes are involved in the PI3K-AKT pathway. CDK1, CCND1, RAF1, CDKN1B and BTRC were defined as the top 5 hub target genes, and patients with high expression of CDK1 showed poor prognosis. Compared with control group, 2.5 µM arecoline treatment increased the proliferation and migration ability of the HepG2 cells. Treatment with 2.5 µM arecoline increased the levels of miR-21-3p, miR-21-5p and miR-1267, upregulated the expression of PI3K-AKT pathway factors, CDK1, CCND1 but decreased RAF1 expression.

Conclusion: A low concentration arecoline can induce the proliferation and migration of HepG2 cells, with the potential mechanism of action linked to high levels of exosomal miR-21 and miR-1267, activation of the PI3K-AKT pathway, upregulation of CDK1 and CCND1, and downregulation of RAF1.

Keywords: Arecoline; CDK1; Cell migration; Cell proliferation; Hepatocellular cancer; PI3K/AKT/mTOR pathway.

Fig. 1

Expression, GO and KEGG analysis of DE-miRNAs in arecoline-induced cancer. A Volcanic plot of the GSE45238 data comparing patients with arecoline-induced cancer and healthy controls. Significantly upregulated miRNAs are indicated with red dotes, while downregulated miRNAs are indicated with green dotes. The dashed line illustrates the cut-off for significance (p < 0.01; T-test). B Circular cluster map. Upregulated miRNAs are indicated with red dotes, while downregulated miRNAs are indicated with green dotes. C Heat map of the top 15 up- or downregulated miRNAs. Red markers upregulation; blue, downregulation. D Enrichment of DE-miRNAs in GO biological processes (BPs). E Enrichment of DE-miRNAs in GO molecular functions (MFs). F Enrichment of DE-miRNAs in GO cellular components (CCs). G KEGG pathway enrichment analysis of DE-miRNAs. Node color and size reflect the log₁₀ (p value) and count, respectively

Fig. 2

Protein–Protein interaction network and prediction of hub genes targeted by DE-miRNAs. A protein interaction network of all predicted DE-miRNA target genes. B protein interaction network of hub-target gene, with redder color indicating with higher degree of interaction. C Histogram of GO analysis of the identified hub-target genes. D Network of GO analysis classified by GO terms. E Network of GO analysis classified by p value. F GO chord of the identified hub-target genes

Fig. 3

Effects of hub gene expression on prognosis of patients with hepatocellular cancer. Expression of the top 5 hub genes was assessed: CDK1, CCND1, RAF1, CDKN1B and BTRC. A Overall survival (OS) of patients showing high and low expression of CDK1. B Disease-free survival (DFS)of patients showing high and low expression of CDK1. C Expression of CDK1 in patients with hepatocellular cancer and healthy volunteers, *p < 0.001. D Expression of CDK1 in patients with hepatocellular cancer at different stages. E OS and DFS in patients showing high or low expression of CCND1. F OS and DFS in patients showing high or low expression of RAF1. G OS and DFS in patients showing high or low expression of CDKN1B. H OS and DFS in patients showing high or low expression of BTRC

Fig. 4

Arecoline treatment affects the cell viability and ultrastructure of HepG2 cells. A Changes in viability of HepG2 hepatoma cells treated with the indicated concentration of arecoline. B-F Quantification of the changes in HepG2 cell viability after treatment with the indicated concentration of arecoline on 1st day B, 2nd day C, 3rd day D, 5th day E and 7th day F. G Transmission electron microscopy images highlighting the ultrastructural changes in HepG2 hepatoma cells treated the indicated concentrations of arecoline. Vs 1st d, ^&p < 0.05; vs 2nd d, ^#p < 0.05; vs 3rd d, ^%p < 0.05; vs 5th d, ^$p < 0.05. Vs 0 μM treatment group, ^ap < 0.05; vs 2.5 μM treatment group, ^bp < 0.05

Fig. 5

Arecoline treatment affects migration and invasion by HepG2 hepatoma cells. A Changes in scratch distance in HepG2 hepatoma cells treated with the indicated concentrations of arecoline. B Changes in migration area in HepG2 hepatoma cells treated with the indicated concentrations of arecoline. C Changes in migration rate in HepG2 hepatoma cells treated with the indicated concentrations of arecoline. D Changes in invasion ability in HepG2 hepatoma cells treated with the indicated concentrations of arecoline, as assessed in a transwell assay. E–F Migration and invasion ability of HepG2 hepatoma cells treated with the indicated concentrations of arecoline, as assessed in a wound healing Vs 0d, ^ap < 0.05; vs 1d, ^bp < 0.05; vs 3d, ^cp < 0.05

Fig. 6

Arecoline treatment promotes cell cycle changes and affects apoptosis in HepG2 hepatoma cells. A-C Fraction of HepG2 hepatoma cells in the G1 A, S B, or G2 C phase after treatment with the indicated concentrations of arecoline. D Quantification of apoptosis in HepG2 cells. E Cell cycle in HepG2 cells on day 1, 3, 7 and con group. F Number of apoptotic cells after treatment with the indicated concentrations of arecoline, as assessed by annexin staining. Vs CON group, ^ap < 0.05; vs 1st day, ^bp < 0.05; vs3rd day, ^cp < 0.05

Fig. 7

Arecoline treatment changes the expression of miRNAs and inflammatory factors via PI3K/AKT/mTOR pathwayin HepG2 hepatoma cells. All analyses were carried out with culture medium of HepG2 hepatoma cells treated with 2.5 μM arecoline and con group. A Exosomal miR-1267 expression. B Exosomal miR-21-3p expression. C Exosomal miR-21-5p expression. D IL-1β protein levels. E TNF-α protein levels. F Relative expression of mRNAs encoding PI3K/AKT/mTOR signaling proteins.G Western blot analysis of PI3K/AKT/mTOR signaling proteins. H Relative expression of PI3K/AKT/mTOR signaling proteins. I Western blot analysis of hub genes. J Relative expression of CDK1, CCND1, RAF1, CDKN1B and BTRC mRNA and protein. Vs CON group, ^ap < 0.05; vs 2.5 μM group, ^bp < 0.05; 5 μM group, ^cp < 0.05