Effect of low dose inorganic arsenic on expression of Sirt1 and cell cycle related genes in L-02 cells

Abstract

Objective To investigate the effect of low dose arsenic on expression of Sirt1 and cell cycle related genes in human normal hepatocytes (L-02). Methods After L-02 cells were treated with 0.0 (control), 0.1, 0.2 and 0.4 μmol/L As2O3 for 24, 48 and 72 h, the effects of arsenic on cell proliferation were detected by CCK-8 assay, the effects of arsenic on Sirt1 protein expression and the intracellular distribution of Sirt1 protein were detected by Western blot, and the effects of arsenic on the expression of Sirt1, p16, p21 and p53 mRNA were detected by quantitative real-time PCR (qRT-PCR). In parallel, the effects of arsenic on acetylation of H3K9, H3K27 and the enrichment degree of Sirt1 in the promoter region of the cell cycle related genes were detected by chromatin immunoprecipitation (ChIP). Results ①Treatment of different doses of arsenic on L-02 cells for 24, 48 and 72 h promoted the proliferation of cells up to 15% to 40% (all P < 0.05). ②After arsenic exposure for 48 h, compared with the control group (1.00 ± 0.04, 1.00 ± 0.03, 1.00 ± 0.06), the expression of p16, p21 and p53 mRNA were down regulated in 0.1 μmol/L arsenic treated group (0.80 ± 0.10, 0.88 ± 0.02, 0.85 ± 0.05, t=-3.22,-5.76,-3.23, all P <0.05); compared with the control group, acetylation of H3K9 and H3K27 of p16, p21 and p53 genes in 0.1 μmol/L arsenic treated group were down regulated (t=-2.89,-9.50,-17.64,-2.88,-3.41, -3.73, all P < 0.05), and the reduction amplitude was about 20% to 70%. ③ After arsenic exposure for 48 h, compared with the control group (1.00 ± 0.01), the intracellular distribution of Sirt1 protein was up regulated in 0.1 μmol/L arsenic treated group (1.04 ± 0.02, t= 3.10, P < 0.05), with an average increase of 4%; the enrichment degree of Sirt1 in the promoter region of p16 and p53 of 0.1 μmol/L arsenic treated group (1.33 ± 0.04, 4.28 ± 0.12) was significantly higher than those in the control group (1.00 ± 0.13, 1.00 ± 0.13, t= 4.20, 32.11, all P < 0.05), with an increase of at least 30%. Conclusion Low-dose of inorganic arsenic could inhibit the expression of cell cycle related genes p16, p21 and p53 and reduce the accumulation of H3K9 and H3K27 acetylation by promoting the Sirt1 nuclear distribution, thereby promoting cell proliferation. Key words: Arsenicals; Epigenetics; Sirt1; Histones; Acetylation; Cell cycle