Effect of microRNA-146a on H2O2-induced oxidative stress activates in AGS gastric cancer cells

Abstract

Objective To observe the concentration and time damage resulting from hydrogen peroxide on human gastric cancer cell line AGS and to investigate the effect regulated from microRNA-146a by constructing the cell model of stress ulcer. Methods (1)The AGS cells were divided into 5 groups, with 4 wells in each group. The content of malondialdehyde and the vigor of superoxide dismutase(SOD) were tested which were kind of lipid peroxidation products from AGS cells that stimulus with 200 μmol/L hydrogen peroxide concentration at time points of 3, 6, 12 and 24 h. (2)The AGS cells were divided into 6 groups, with 4 wells in each group. Then the content of malondialdehyde and the vigor of SOD were tested which were kind of lipid peroxidation products from AGS cells that stimulus with 50, 100, 200, 400 and 600 μmol/L hydrogen peroxide concentration all after 6 hours. (3) The AGS cells were divided into 2 groups, with 4 wells in each group. Then the AGS cells were excited 6 hours with 200 μmol/L hydrogen peroxide applying real-time PCR to detect gene expression of miRNA-146a of AGS cells. (4) The AGS cells were divided into 4 groups, with 4 wells in each group. Cells in miRNA-146a control group were transfected with microRNA control; cells in miRNA-146a enhancement group were transfected with miRNA-146a mimics; cells in miRNA-146a inhibition group were transfected with miRNA-146a inhibitor. After cultivating for 24 hours, miRNA-146a expression, the content of malondialdehyde and the vigor of SOD of the cells wer tested that have been stimulated 6 hours by hydrogen peroxide. Statistics were chose one-factor analysis of variance between groups and LSD-t test for the two groups. Results (1)After 200 μmol/L hydrogen peroxide stimulation, comparing the malondialdehyde content and SOD vitality in cells between untreated group and different time training group, the differences were statistically significant(F=46.744, 42.736, P values were less than 0.01). As the extension of the time, the malondialdehyde content of cells was gradually increased, and peaked at(2.15±0.50)μmol/mg in the culturing 6 h, then gradually reduced. Contrarily, as the extension of the time, the SOD vitality of cells was gradually reduced, and footed at(6.51±0.54)μmol/mg in the culturing 6 h, then gradually increased. (2) After 6 hours treatment of different concentration hydrogen peroxide, comparing the malondialdehyde content and SOD vitality in cells between untreated group and different time training group, the differences were statistically significant(F=152.786, 129.231, P values were less than 0.01). With the increase of concentration, malondialdehyde content gradually increased, and highest at (6.51±0.54)U/mg dealing with 600 μmol/L treatment group, while the SOD vitality gradually decreased, and lowest at (3.14±0.10) U/mg dealing with 600 μmol/L treatment group. (3)The miRNA-146a expression of AGS cells of the experimental group was significantly higher than the normal group (t=-17.398, P<0.01). (4) After 6 hours stimulation of 200 μmol/L hydrogen peroxide, comparing the relative expression of miRNA-146a, malondialdehyde content and SOD vitality in cells among miRNA-146a enhancement group, untreated group, miRNA-146a control group and miRNA-146a inhibitor group, the differences were statistically significant (F=232.643, 1 070.580, 56.191, P values were less than 0.01). The miRNA-146a expression of the miRNA-146a enhancement group was hundreds of times than others, the differences were statistically significant(P values were less than 0.01), while the SOD vitality of the miRNA-146a enhancement group was higher than the miRNA-146a control group and the miRNA-146a inhibition group, the differences were statistically significant(P values were less than 0.01), at the same time, the malondialdehyde content of the miRNA-146a enhancement group was lower than the miRNA-146a control group and the miRNA-146a inhibition group, the differences were statistically significant(P values were less than 0.01). Conclusions Applying 200 μmol/L hydrogen peroxide to deal with AGS cells for 6 hours can better construct the cell model of stress ulcer. miRNA-146a can negatively regulate oxidative stress reaction of the AGS cells stress ulcer resulting from hydrogen peroxide. Key words: Stress ulcer; Reactive oxygen species; Hydrogen peroxide; Cells model; MicroRNAs