Corylin Suppresses Hepatocellular Carcinoma Progression via the Inhibition of Epithelial-Mesenchymal Transition, Mediated by Long Noncoding RNA GAS5

Abstract

Corylin is a flavonoid extracted from the nuts of Psoralea corylifolia L. (Fabaceae), which is a widely used anti-inflammatory and anticancer herb in China. Recent studies revealed antioxidant, anti-inflammatory, and bone differentiation-promoting effects of corylin. However, there are no studies examining the anticancer activity of corylin. In this study, we used cells and animal models to examine the antitumor effects of corylin on hepatocellular carcinoma (HCC) and then studied its downstream regulatory mechanisms. The results showed that corylin significantly inhibited the proliferation, migration, and invasiveness of HCC cells and suppressed epithelial-mesenchymal transition. We found that the anti-HCC mechanism of corylin's action lies in the upregulation of tumor suppressor long noncoding RNA growth arrest-specific transcript 5 (GAS5) and the activation of its downstream anticancer pathways. In animal experiments, we also found that corylin can significantly inhibit tumor growth without significant physiological toxicity. The above results suggest that corylin has anti-HCC effects and good potential as a clinical treatment.

Keywords: corylin; epithelial-mesenchymal transition; hepatocellular carcinoma; lncRNA GAS5.

Figure 1

Corylin inhibits the proliferation, migration, and invasiveness of hepatocellular carcinoma (HCC) cells. (A) The cell proliferation capacities of Huh7 and HepG2 were monitored at the indicated time points using an xCELLigence real-time cell analyzer. Corylin significantly inhibited the proliferative capacities of both cell lines. p < 0.05 (*), p < 0.01 (**). Data are expressed as the mean ± S.D. of three independent experiments; (B) Wound-healing abilities were compared between corylin- and vehicle-treated Huh7 cells (left panel). Corylin reduced the wound-healing ability of Huh7 cells. The quantitative wound-healing assay results are shown in the right panel. p < 0.001 (***). Magnification: 100×; (C) Cell migration capacity was compared between Huh7 and HepG2 cells treated with/without corylin using a Transwell assay (left panel). Corylin significantly reduced cell migratory ability in both cell lines. Quantitative cell migration assay results are shown in the right panel; (D) Invasion assays were performed using Matrigel-coated polyethylene terephthalate membrane inserts. Corylin significantly inhibited cell invasion ability in both cell lines at a concentration of 30 μM (left panel). Quantitative cell invasion assay results are shown in the right panel. All experiments were performed in triplicate. Mock: cells treated with Dulbecco’s modified Eagle medium (DMEM). Vehicle: cells treated with dimethyl sulfoxide (DMSO). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). Magnification: 100×.

Figure 2

Corylin suppressed the migration and invasion capacities of HCC cell via inhibiting epithelial-mesenchymal transition (EMT). (A) The expression of EMT-related proteins in Huh7 and HepG2 cells after treatment with corylin or vehicle were analyzed by Western blotting. β-actin served as an internal control. A dose of 30 μM corylin significantly reduced the expression of EMT-related proteins in both cells. Densitometric analyses are shown in (B). The histogram shows the relative expression level of EMT-related proteins in the corylin-treatment compared to the mock-treatment group. Data are expressed as the mean ± S.D. of three independent experiments. * p < 0.05, *** p < 0.001, when compared to vehicle control. Mock: cells treated with DMEM medium. Vehicle: cells treated with DMSO.

Figure 3

Corylin suppresses tumor growth in mice. (A,B) Huh7 cells (5 × 10⁶) were implanted into nude mice (n = 5). Representative images show the tumor xenografts at 35 days after implantation. Corylin (60 mg/kg, IP) significantly reduced tumor growth. IP: intraperitoneal injection; (C) Tumor volumes were measured every three days after implantation, and the volume of each tumor was calculated (length × width² × 0.5). Bars indicate S.D. *** p < 0.001; (D) Body weights were calculated every three days after implantation. Mouse body weights in all groups did not significantly differ; (E) Immunohistochemical staining showed that corylin reduced EMT-related protein expression levels. Magnification: 400×. Scale bar = 100 μm.

Figure 4

Corylin affected the expression of genes associated with cell growth and apoptosis pathways. (A) Heatmap comparing significant differentially expressed lncRNAs in Huh7 and HepG2 cells treated with or without corylin. Gene expression was significantly changed compared to that of the control group after corylin treatment. Bar charts represent the enriched biological processes (B) and biological pathways (C) associated with the differentially expressed genes after corylin treatment. Corylin affected the expression of genes associated with cell growth and apoptosis pathways.

Figure 5

Corylin increases the expression of long noncoding RNA GAS5 in HCC cell lines. (A) The whole-transcriptome sequencing data revealed that 77 long noncoding RNAs (lncRNAs) manifested more than a 2-fold change in expression after corylin treatment in both cell lines; (B) Huh7 and HepG2 cells were treated with corylin for 48 h, and GAS5 expression was analyzed by quantitative real-time RT-PCR, which showed that 30 μM corylin significantly induced GAS5 expression. *** p < 0.001; (C) Representative results of the in situ hybridization of lncRNA GAS5 in the xenografts of mice treated with corylin or vehicle. Corylin significantly induced GAS5 expression. Scale bar = 100 μm. Error bars indicate S.D.

Figure 6

Corylin inhibits the proliferation, migration, and invasion of HCC cells by inducing lncRNA GAS5. (A–C) The inhibitory effects of corylin on cell proliferation, migration, and invasion were significantly reversed by treatment with GAS5 siRNA in the Huh7 and HepG2 cells. Magnification: 100×. Quantitative cell migration and invasion assay results are shown in (D,E). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). Error bars indicate S.D.