Effect And Mechanism Of S100A4 On Ferroptosis Induced By Erastin In Gastric Cancer Cells

Introduction:Gastric cancer is a common malignant tumor,which is seriously harmful to human health.The early diagnosis rate of gastric cancer is low and lack of specificity.Usually,patients with gastric cancer are in the middle and late stage when they are diagnosed.Although surgery,chemotherapy,immunotherapy and targeted therapy can be used to improve the survival rate of some patients with gastric cancer.However,due to the complex pathogenesis and strong heterogeneity of gastric cancer,the therapeutic effect and overall prognosis of patients are still not optimistic,so there is an urgent need to find new adjuvant treatments to improve clinical efficacy.Ferroptosis is a newly discovered iron-dependent regulatory mode of cell death in recent years,which is caused by the accumulation of toxicity of lipid peroxidation on the cell membrane.It is different from apoptosis,necrosis and other types of cell death in morphology,genetics and molecular biology.More and more studies have shown that ferroptosis is closely related to a variety of human diseases and plays an important role in the occurrence and development of tumors.Therefore,inducing ferroptosis in tumor cells may represent a new strategy of anti-tumor therapy,and drugs or compounds that induce ferroptosis in tumor cells are also considered as potential drugs for tumor therapy.Erastin is one of the most commonly used inducers of ferroptosis.By inhibiting cystine uptake by cells,it leads to a decrease in GSH synthesis and lipid peroxidation,resulting in ferroptosis.Some studies have shown that erastin can induce ferroptosis in a variety of tumor cells,including breast cancer cells,prostate cancer cells and gastric cancer cells.Previous studies in our team have shown that erastin can induce ferroptosis in gastric cancer cell MGC803,and knocking down GDF15 can enhance erastin-induced ferroptosis in MGC803 cells,but the mechanism of the effect of erastin on ferroptosis in gastric cancer cells remains to be further studied.S100A4,also known as PEL98,P9KA,FSP1,etc.,is a member of the S100 family of calcium binding proteins.Many studies have shown that S100A4 is highly expressed in a variety of human malignant tumors,such as gastric cancer,lung cancer,prostate cancer and colorectal cancer,and is closely related to tumor metastasis and poor prognosis.Many studies have confirmed that S100A4 affects the proliferation,migration,apoptosis and autophagy of many kinds of tumor cells,such as gastric cancer,lung cancer and so on.Thus it can be seen that S100A4 plays a very important role in the occurrence and development of tumors such as gastric cancer by affecting the biological characteristics of tumor cells,but the relationship between S100A4 and ferroptosis of tumor cells has not been reported.Our team used chip technology to analyze the differential gene expression profile of gastric cancer cell MGC803 after S100A4 knockdown.We found that some of the differential genes were related to ferroptosis.It is speculated that S100A4 may play an important role in the regulation of ferroptosis in gastric cancer cells.In order to verify the above conjecture,we plan to carry out relevant experimental research.We first verified that ferroptosis inducer erastin could induce ferroptosis in gastric cancer cells,then explored the effect of erastin on the expression of S100A4 in gastric cancer cells,and then observed the effect of transfection of S100A4 expression vector on ferroptosis induced by erastin in gastric cancer cells.In order to further explore the role of S100A4 in ferroptosis of gastric cancer cells,we focus on the effects of knock-down S100A4 combined with low concentration erastin on ferroptosis of gastric cancer cells.On this basis,we intend to detect the levels of ferroptosis-related indexes lipid ROS and MDA,as well as the expression of GPX4,a key antioxidant gene regulating ferroptosis,and preliminarily understand the mechanism of the effect of knock-down S100A4 combined with low concentration erastin on ferroptosis of gastric cancer cells.In this study,gastric cancer was taken as the research object to explore the relationship between S100A4 and ferroptosis for the first time.Through this study,we hope to clarify the role of S100A4 in erastin-induced ferroptosis in gastric cancer cells,and preliminarily understand its mechanism,so as to provide new ideas and theoretical basis for the development of new strategies for the treatment of gastric cancer,and will also have implications for other tumors.Materials and methods:1.Experimental materials:Human gastric cell line MGC803,human gastric cell line HGC27.2.Experimental methods:（1）Cell culture:Human gastric cancer cell line MGC803 and human gastric cancer cell line HGC27 were cultured in 5%CO₂and 37℃cell incubator.（2）Transient transfection:GV712-S100A4 expression vector and S100A4-si RNA were transfected into MGC803 cells and S100A4-si RNA was transfected in HGC27 cells for subsequent experiments.（3）Real-time quantitative PCR（q RT-PCR）:Detect the m RNA expression level of S100A4in MGC803 cells and HGC27 cells and the m RNA expression level of GPX4 in MGC803cells.（4）Western blotting（WB）:Detect the protein expression level of S100A4 in MGC803cells and HGC27 cells and the protein expression level of GPX4 in MGC803 cells.（5）CCK8 experiment:Detect the viability of MGC803 cells and HGC27 cells.（6）Flow cytometry:Detect the content of lipid reactive oxygen species in MGC803 cells and HGC27 cells.（7）Malondialdehyde（MDA）detection experiment:Detect the content of malondialdehyde in MGC803 cells and HGC27 cells.（8）Transmission electron microscope:Observe the morphology of cell mitochondria.Results:1.Effect of erastin treatment on ferroptosis of gastric cancer cells MGC803 and HGC27.（1）Effect of erastin on ferroptosis of MGC803 cells:CCK8 results showed that erastin induced MGC803 cell death in a dose-dependent manner（P<0.05）,and ferroptosis inhibitor ferrostatin-1 or liproxstatin-1 could save erastin-induced cell death（P<0.05）.Transmission electron microscope showed that mitochondrial morphology changed,mitochondrial volume became smaller and cristae decreased or even disappeared in MGC803 cells treated with erastin.（2）Effect of erastin on ferroptosis of HGC27 cells:CCK8 results showed that erastin induced HGC27 cell death in a dose-dependent manner（P<0.05）,and ferroptosis inhibitor ferrostatin-1 or liproxstatin-1 could save erastin-induced cell death（P<0.05）.2.Effect of erastin treatment on S100A4 expression in gastric cancer cells.q RT-PCR and WB results showed that erastin could down-regulate the expression of S100A4 m RNA and protein in gastric cancer cells MGC803 and HGC27（P<0.05）.3.Effect of S100A4 overexpression on ferroptosis induced by erastin in gastric cancer cell line MGC803.（1）WB results showed that overexpression of S100A4 could reverse the decreased expression of S100A4 protein induced by erastin treatment（P<0.05）.（2）CCK8 results showed that overexpression of S100A4 could save the decrease of MGC803 cell viability induced by erastin treatment（P<0.05）.4.Effect of knocking down S100A4 on ferroptosis of gastric cancer cells induced by erastin.（1）The effect of knocking down S100A4 on erastin-induced ferroptosis in MGC803 cells:CCK8 results showed that erastin at the concentration of 10μM,20μM and 25μM could significantly inhibit the cell viability at the time point of 24 h,and the cell viability could be further inhibited by combined knockdown of S100A4（P<0.05）.At the time point of48 h treatment,the erastin of 2.5μM and 5μM had no significant effect on the cell viability,but the combined knockdown of S100A4 could significantly inhibit the cell viability（P<0.05）.CCK8 results showed that ferroptosis inhibitor ferrostatin-1 or liproxstatin-1 could save the decrease of MGC803 and HGC27 cell viability induced by knocking down S100A4 combined with low concentration erastin（2.5μM）treatment（P<0.05）.Transmission electron microscope results showed that the morphology of mitochondria was seriously damaged in MGC803 cells treated with low S100A4combined with low concentration of erastin.（2）The effect of knocking down S100A4 on erastin-induced ferroptosis in HGC27 cells:CCK8 results showed that erastin of 2.5μM and 5μM could significantly inhibit cell viability after 24 h treatment,and combined knockdown of S100A4 could further inhibit cell viability（P<0.05）.Eastin at the concentration of 1μM had no significant effect on cell viability,but combined knockdown of S100A4 could significantly inhibit cell viability（P<0.05）.CCK8 results showed that ferroptosis inhibitor ferrostatin-1 or liproxstatin-1 could save the decrease of HGC27 cell viability induced by knocking down S100A4 combined with low concentration erastin（1μM）treatment（P<0.05）.5.Preliminary study on the mechanism of the effect of knocking down S100A4 combined with low concentration of erastin on ferroptosis in gastric cancer cells.（1）Flow cytometry results showed that knockdown S100A4 combined with low concentration of erastin could significantly increase the content of lipid ROS in MGC803and HGC27 of gastric cancer cells（P<0.05）.（2）Malondialdehyde detection results showed that knockdown S100A4 combined with low concentration erastin could significantly increase the content of malondialdehyde in MGC803 and HGC27 gastric cancer cells（P<0.05）.（3）qRT-PCR and WB results showed that knockdown S100A4 combined with low concentration of erastin could significantly inhibit the m RNA and protein expression of GPX4 in gastric cancer cell line MGC803（P<0.05）.Conclusions:1.Down-regulation of S100A4 expression can mediate erastin-induced ferroptosis in gastric cancer cells.2.Inhibition of S100A4 expression could increase the sensitivity of gastric cancer cells to ferroptosis induced by erastin.3.Knocking down S100A4 at low concentration of erastin may promote ferroptosis of gastric cancer cell line MGC803 by inhibiting the expression of GPX4.