| Background: Lung cancer(LCA),the number one malignancy worldwide,is the leading cause of tumor-related deaths worldwide,and its incidence has steadily increased over the past decades.According to recent studies,the prognosis and five-year survival rates of lung cancer patients are extremely low and have also shown a trend toward younger age in recent years.Among the pathological staging of lung cancer,Non-small cell carcinoma(NSCLC)subtype accounts for the largest proportion of all lung cancers,about 85%,and Lung adenocarcinoma(LUAD)is the most prevalent among patients with non-small cell lung cancer.Metformin is a first-line drug for type 2diabetes,and its pharmaceutical principle is to control the blood glucose level of patients by enhancing insulin sensitivity.In recent years,more and more studies have shown that metformin not only has the effect of reducing blood glucose,but also has a potential anti-tumor effect.A large number of studies have confirmed that metformin can effectively inhibit the growth activity of a variety of cancer cells,but its mechanism has not been reached consensus.Ferroptosis,an iron-dependent programmed necrosis,is now widely recognized as a key factor influencing the development and progression of many cancers.The main mechanism may be the regulation of ferroptosis by modulating the classical ferroptosis signaling pathway x CT-GPX4,while regulating the levels of Glutathione(GSH),intracellular Reactive oxygen species(ROS),etc.Objective:With the rapid development of medical research and medical technology,the early diagnosis and treatment of lung cancer have been significantly improved,but the prognosis of lung cancer patients is still poor,and expensive chemotherapeutic drugs have brought considerable economic pressure to patients.Therefore,it is urgent to find an affordable drug that can effectively act on the target site.In this study,the expression levels of the ferroptosis key molecules SLC7A11 and GPX4 in various tumor tissues including lung adenocarcinoma were firstly pre-explored,and finally whether metformin could inhibit the activity of lung adenocarcinoma cells by regulating SLC7A11 and GPX4 was investigated.Methods: 1,TCGA and TIMER database was used to explore the the expression of ferroptosis target molecules SLC7A11 and GPX4 in pan-cancer.2,Bioinformatics methods were used to compare the expression differences of the ferroptosis target molecules SLC7A11 and GPX4 in serum of patients with lung adenocarcinoma and control one and the effects on the survival rate.3,The morphological changes of A549 lung adenocarcinoma cells in each experimental group(0,10,20,40 mmol/L)were observed using inverted phase contrast microscope.4,The CCK-8 assay was used to determine the changes in cell viability of lung adenocarcinoma A549 cells after 24-hour treatment with different drug concentrations(0,5,10,20,30,and 40mmol/L),in order to confirm whether metformin inhibits the viability of lung adenocarcinoma A549 cells.5,The levels of GSH in A549 lung adenocarcinoma cells of each experimental group(0,10,20,40 mmol/L)were detected using GSH kit.6,The ROS level of A549 cells in each experimental group(0,10,20,40 mmol/L)was detected using DCFH-DA probe(flow cytometry).7,The expression of SLC7A11 and GPX4 in A549 cells of different experimental groups(0,10,20,40 mmol/L)was detected using Western blot method(immunoblotting technique).Results: 1,The application of bioinformatics research has revealed that SLC7A11 and GPX4 show high expression in various tumor tissues.While in lung adenocarcinoma and normal population,the expression differences of SLC7A11 and GPX4 are larger.At the same time,this study also found that the survival rate of patients with lung adenocarcinoma with high expression of SLC7A11 and GPX4 genes was significantly lower than that of patients with low expression.This suggested that lung adenocarcinoma might have ferroptosis resistance.2,The A549 cells of lung adenocarcinoma treated with different concentrations of metformin were observed under a phase contrast inverted microscope.Compared with the control group,the cells grew in poor shape,with irregular shape,smaller shrinkage and discrete appearance,and the number in the field of view was significantly reduced.3,The results of CCK8 assay showed that metformin had a significant inhibitory effect on lung adenocarcinoma A549 cells,and the inhibitory effect was significantly and positively correlated with the concentration.4,After treatment with different concentrations of metformin in A549 cells of lung adenocarcinoma in each experimental group,GSH levels in each group decreased with the increase of drug concentration.The relative contents of GSH production in each experimental group(0,10,20,and 40mmol/L)were 1.00±0.00,0.96±0.00,0.94±0.00 and 0.65±0.00,respectively.5,After treatment with different concentrations of metformin in each experimental group of lung adenocarcinoma A549 cells,the amount of ROS produced by each group increased with the increase of drug concentration.The relative contents of ROS production in each experimental group(0,10,20,and 40mmol/L)were 1.00± 0.00,5.06±0.93,6.92±0.95,and 31.63±6.07,respectively.6,Western blot analysis showed that the expression level of the target protein SLC7A11 decreased with the increase of the metformin concentration,and the expression levels of SLC7A11 in the low,medium and high dose groups were different from those in the control group(P<0.05).However,only the expression of GPX4 protein in the high-dose group was statistically significant from that in the control group(P<0.05).These results suggested that high dose of metformin might inhibit the expression of SLC7A11 and GPX4.Conclusion: This study found that metformin can promote the production of ROS and inhibit the production of GSH,which inhibited the proliferation activity of lung adenocarcinoma cells,and the expression of SLC7A11 and GPX4 proteins,the key molecules of ferroptosis in lung adenocarcinoma A549 cells treated with metformin,decreased.Therefore,we suggest that metformin may inhibit the activity of lung adenocarcinoma cells by down-regulating ferroptosis molecules SLC7A11 and GPX4. |
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