Effect Of Silencing SLC1A5 Gene On MDA,the Final Product Of Ferroptosis Lipid Peroxidation In Gastric Cancer AGS Cells

Objective:To investigate the expression of solute carrier family 1 member 5（SLC1A5）in gastric cancer and its effect on malondialdehyde（MDA）,the end product of ferroptosis lipid peroxidation.Methods:（1）The expression levels of SLC1A5 in gastric cancer tissues and normal gastric mucosal tissues were analyzed using the gene expression profiling interactive analysis2（gepia2）database based on the Cancer Genome Atlas（TCGA）database and the genotype tissue expression（GTEX）database.Then the survival analysis was performed and Kaplan Meier（K-M）survival curves were plotted based on clinical data of gastric cancer patients from TCGA database.（2）Small interfering Ribonucleic Acid（si RNA）targeting SLC1A5messenger RNA（m RNA）（SLC1A5-si RNA）was designed and synthesized.The m RNA level of SLC1A5 in Human gastric adenocarcinoma cells（AGS）was silenced by SLC1A5-si RNA transfection.Blank control group and negative control（control with negative sequence,NC）group were set.The m RNA levels of SLC1A5 were determined by real-time quantitative polymerase chain reaction（RT-q PCR）,The relative m RNA expression was calculated by the2^-ΔΔCtmethod.（3）The expression of SLC1A5 protein in gastric carcinoma and normal gastric mucosa was determined by the University of Alabama at Birmingham cancer data analysis portal（ualcan）database.Confirmation of SLC1A5 protein in AGS cells by Western blot（WB）in each group.（4）Thiobarbital acid（TBA）method was used to detect MDA,the end product of lipid peroxidation in AGS cells,and the change of MDA content in AGS cells after silencing SLC1A5.Results:（1）the results of gepia2 analysis indicated that SLC1A5 was increased in gastric cancer tissues compared with normal gastric mucosa（p<0.05）.（2）K-M survival curve results indicated that the 5-year overall survival rate of the SLC1A5 high expression group was higher than that of the SLC1A5 low expression group,but the overall survival rate of the SLC1A5 high expression group was lower than that of the SLC1A5 low expression group from the 6th year,the hazard ratio（HR）=0.81 for the high expression of SLC1A5;The disease-free survival rate was higher in the group with high expression of SLC1A5 than that in the group with low expression of SLC1A5,HR=0.86.（3）Comparison of the relative expression of SLC1A5 m RNA 24 hours after transfection of AGS cells in each group:The SLC1A5-si RNA group was significantly lower than the blank control group（p<0.01）,with a statistically significant difference;The SLC1A5 si RNA group was lower than the blank control group（p<0.05）;There was no significant change in the NC group compared to the blank control group（p>0.05）,and the result was not statistically significant.（4）Comparison of the relative expression of SLC1A5 protein 48 hours after transfection:The SLC1A5 si RNA group was significantly lower than the blank control group（p<0.01）,with a statistically significant difference;The SLC1A5 si RNA group was lower than the blank control group（p<0.05）;There was no significant change in the NC group compared to the blank control group（p>0.05）,and the result was not statistically significant.（5）At 48 hours after transfection,the relative content of MDA in the SLC1A5-si RNA group was significantly lower than that in the blank control group（p<0.0001）;The relative content of MDA in the SLC1A5-si RNA group was significantly lower than that in the NC group（p<0.0001）;There was no significant change in the NC group compared to the blank control group（p>0.05）,and the result was not statistically significant.Conclusion:（1）SLC1A5 was increased in gastric cancer tissues compared with normal gastric mucosa.High expression of SLC1A5 may lead to better prognosis than low SLC1A5,and high expression of SLC1A5 can reduce the risk of death in patients with gastric cancer.（2）The interference sequence we designed can successfully silence the expression of SLC1A5m RNA and protein after si RNA transfection.After silencing the SLC1A5 gene,the content of MDA in AGS cells decreased under the condition of unit mass protein,indicating that silencing SLC1A5 can inhibit the occurrence of ferroptosis and lipid peroxidation in gastric cancer cells,which provides a good starting point for further exploring the mechanism of ferroptosis in gastric cancer.