DNA methylation and expression changes of ferroptosis related genes SLC7A11 and CDKN1A in human hepatic stellate cell activation induced by sodium arsenite

Background The accumulation of inorganic arsenic in the liver can lead to liver fibrosis. Solute carrier family 7 member 11 (SLC7A11) and cyclin dependent kinase inhibitor 1A (CDKN1A) are ferroptosis related genes that are abnormally expressed in liver and fibrosis diseases, but their impacts on inorganic arsenic induced liver fibrosis has not been reported yet.

Objective To explore the effect of different doses of sodium arsenite on the migration ability of human hepatic stellate (LX-2) cells and the changes in DNA methylation and expression of SLC7A11 and CDKN1A in LX-2 cell activation induced by sodium arsenite.

Methods LX-2 cells were treated with different doses of sodium arsenite for 24 h. The experiment set up four groups: control group (0 μmol·L⁻¹), low-dose group (5 μmol·L⁻¹), medium-dose group (10 μmol·L⁻¹), and high-dose group (15 μm·L⁻¹). Possible morphological changes of LX-2 cells were observed under an inverted microscope in each group. Cell migration and movement were determined by cell scratch assay. Mitochondrial morphology was observed in the control and the high-dose groups under a transmission electron microscope (TEM). Fe²⁺ fluorescence intensity was detected with a FerroOrange fluorescence probe. Differentially methylated sites were screened using the Illumina 850 K methylation chip. Ferroptosis related markers (SLC7A11 & CDKN1A) and hepatic stellate cell activation related markers transforming growth factor-β1 (TGF-β1) and collagen type Ⅲ (Collagen Ⅲ) were detected by real-time fluorescence quantitative PCR and Western blot.

Results With the increase of exposure dose, the cells retracted tentacles and gradually became round, and the healing of cell scratches gradually deteriorated. Destruction of mitochondrial membrane integrity and mitochondrial crista fracture in some cells were observed under a TEM in the high-dose group. Laser confocal microscopy showed that the fluorescence intensity of Fe²⁺ gradually increased with the increase of exposure dose. The results of methylation chip displayed that compared with the control group, high methylation was found in the high-dose group at the cg22659014 site in the promoter region of SLC7A11, and at the cg17964532 site in the promoter region of CDKN1A. With the increase of exposure dose, the mRNA and protein expression levels of SLC7A11 decreased (P<0.001); the CDKN1A mRNA expression level did not change significantly between groups (P>0.05), and its protein expression levels in the low-, medium-, and high-dose groups were higher than that in the control group (P<0.001). The TGF-β1 mRNA expression levels in the medium- and high-dose groups were higher than that in the control group (P<0.001), and its protein expression levels increased with the increase of exposure dose (P<0.001). The CollagenⅢ mRNA expression level increased with the increase of exposure dose (P<0.001), and its protein expression level was higher only in high-dose group than that in the control group (P<0.001).

Conclusion Sodium arsenite exposure can weaken the migration ability of LX-2 cells, and the ferroptosis related genes SLC7A11 and CDKN1A are involved in the process of sodium arsenite-induced activation of LX-2 cells.