| Introduction: Gastric cancer is one of the most common malignant tumors in digestive system.Its occurrence and development is a complicated process involving many factors.Because the onset of gastric cancer is relatively insidious,most patients are already in the advanced stage when diagnosed.Although traditional methods such as surgery and chemotherapy have been optimized and improved,and targeted drugs have been applied,the therapeutic effect is still not ideal.It will be of great significance to explore new therapeutic strategies to assist the currently commonly used therapeutic methods to improve the prognosis of patients with gastric cancer.Abnormal energy metabolism is an important characteristic of tumor,which plays a crucial role in tumor occurrence and development.Glutamine is a key nutrient and the most abundant amino acid in the body.It is involved in a variety of biosynthesis and metabolic processes.The energy production of cancer cells is highly dependent on the metabolism of glutamine,and many cancers are particularly dependent on glutamine to maintain the survival and proliferation of cancer cells.Recent studies have shown that the highest uptake of glutamine by cancer cells is determined by their internal programming.The above studies indicate that glutamine is a key nutrient for cell growth and survival and plays a key role in cancer,especially for cancer cells.After in vitro glutamine deprivation treatment,cancer cells will fall into a state of stagnant proliferation or even death.Therefore,targeting glutamine metabolism may be a promising strategy for the treatment of malignant tumors.Studies have shown that glutamine deprivation significantly affects the biological function of tumor cells.Glutamine deprivation in vitro significantly inhibited the proliferation of pancreatic and bladder cancer cells,promoted cell death,and inhibited the invasion and metastasis of ovarian cancer cells.However,it has also been shown that nutritional stress induced by glutamine deprivation can promote epithel-mesenchymal transformation of pancreatic ductal adenocarbines,thereby promoting tumor epithelial cell migration to nutrient-rich areas.Notably,gastric cancer cells showed higher levels of glutamine metabolism.At present,studies on the relationship between gastric cancer and glutamine metabolism mainly focus on the influence of key molecules of glutamine metabolism,such as inhibitors such as glutamine transporters,on the characteristics of gastric cancer cells,such as proliferation,survival,stem cell characteristics,etc.However,there are few reports on the influence of glutamine deprivation on the characteristics of gastric cancer cells,and the mechanism is not clear.In-depth research on this will be of great significance.S100A4 is a member of the calcium ion binding protein S100 family.S100A4 is highly expressed in most tumors,including gastric cancer,and the expression level of S100A4 in tumors is closely related to tumor progression and metastasis,indicating its potential importance in tumor diagnosis and treatment.Previous studies by other scholars and our research group have confirmed that S100A4 has an important effect on the biological characteristics of gastric cancer cells,but the effect of S100A4 on tumors under glutamine deprivation has not been reported.Glucose deprivation has been shown to down-regulate S100A4 expression in prostate cancer cells.We speculated that glutamine deprivation,as a key nutrient component,may also affect the expression of S100A4 in gastric cancer and other tumor cells,and S100A4 may mediate the effects of glutamine deprivation on the biological characteristics of gastric cancer cells.In order to confirm the above speculation,we used relevant molecular biological techniques to detect the effects of glutamine deprivation on the expression level of S100A4 in gastric cancer MGC803 and HGC27 cells and on the biological characteristics of proliferation,migration and apoptosis of gastric cancer cells.Meanwhile,the response experiment was conducted to detect whether S100A4 mediated glutamine deprivation had an effect on biological characteristics of gastric cancer cells,and the molecular mechanism of glutamine deprivation regulating S100A4 expression was preliminarily studied.It provides new ideas and clues for further research on the molecular mechanism of the effect of S100A4 on the biological characteristics of gastric cancer cells under the condition of glutamine deprivation and targeting glutamine metabolism in the treatment of gastric cancer.Materials and Methods:1.Experimental materials: Human gastric cancer MGC803 cell line,human gastric cancer HGC27 cell line and human gastric mucosal epithelium GES-1 cell line.2.Methods:(1)After glutamine deprivation(gradient reduction of glutamine concentration),the mRNA and protein expression of S100A4 were detected by Real-time PCR and Western blot.The proliferative ability of gastric cancer cells was detected by CCK8 cell proliferation assay.According to the above experiment,0.2 m M was selected as the condition of glutamine deprivation for subsequent experiment.Transwell assay and flow cytometry were used to detect the migration ability,cell cycle and apoptosis of gastric cancer cells after glutamine deprivation.(2)Real-time PCR and Western blot were used to detect the effect of transfected S100A4 expression vector on the expression of S100A4 mRNA and protein.Transfected with S100A4 expression vector,a response experiment was conducted under glutamine deprivation condition to preliminarily investigate whether S100A4 mediates glutamine deprivation on the above biological characteristics of gastric cancer cells.(3)Glutamine-deprived gastric cancer cells were treated with actinomycin D,and S100A4 mRNA levels were detected to preliminarily determine whether glutamine deprivation regulates the expression of S100A4 mRNA by affecting its stability.(4)In glutamine deprivation culture,Wnt/β-catenin pathway activator Ch IR-99021 was added to detect β-catenin and S100A4 expression.The effects of glutamine deprivation on β-catenin mRNA and protein expression were also determined.The effect of Wnt/β-catenin pathway activator on β-catenin and S100A4 expression under glutamine deprivation was preliminarily investigated.Results:1.Glutamine deprivation down-regulates the expression of S100A4 in gastric cancer cells.Real-time PCR and Western blot results showed that the mRNA and protein expression levels of S100A4 in MGC803 and HGC27 cells in glutamine deprivation group were significantly lower than those in control group(P < 0.05).2.Glutamine deprivation affects the biological characteristics of gastric cancer cells(1)The results of microscope examination showed that after glutamine deprivation,gastric cancer cells were not full,poor refractive,irregular shape,and the interval between cells increased.(2)The results of CCK8 cell proliferation experiment showed that the proliferation ability of MGC803 and HGC27 cells in glutamine deprivation group was significantly lower than that in control group(P < 0.05).The proliferation ability of gastric mucosal epithelial cells GES-1 in glutamine deprivation group had no significant effect on that in control group.The proliferation capacity of GES-1 in glutamine complete deprivation group was significantly lower than that in control group(P < 0.05).(3)The results of cell cycle experiment showed that the proportion of cells in G2 phase was significantly higher than that in control group,and the proportion of cells in S phase was significantly lower than that in control group(P < 0.05),suggesting that glutamine deprivation could block MGC803 cells in G2 phase.(4)The apoptosis of MGC803 cells in glutamine deprivation group was not significantly different from that in control group(P > 0.05).(5)Transwell experiment showed that the migration ability of MGC803 and HGC27 cells in glutamine deprivation group was significantly lower than that in control group(P< 0.05).3.Transfection of S100A4 expression vector up-regulated the expression of S100A4 in gastric cancer cellsThe expression level of S100A4 in gastric cancer cells was significantly higher than that in control group after transfection with S100A4 expression vector under conventional culture conditions(P < 0.05).After transfection of S100A4 expression vector,the decreased S100A4 expression level caused by glutamine deprivation could be recovered(P < 0.05).4.Effects of S100A4-mediated glutamine deprivation on proliferation and migration of gastric cancer cells(1)The response experiment showed that the proliferation and migration ability of MGC803 and HGC27 cells could be partially restored after transfection of S100A4 expression vector and cultured under glutamine deprivation condition(P < 0.05).(2)The response experiment showed that the cycle arrest of MGC803 cells induced by glutamine deprivation could be partially recovered after transfection of S100A4 expression vector and cultured under glutamine deprivation condition(P < 0.05).5.Preliminary study on the molecular mechanism of glutamine deprivation regulating S100A4 expression in gastric cancer MGC803 cells.(1)Under glutamine deprivation,there was no significant difference in S100A4 mRNA level between actinomycin D treatment group and control group(P > 0.05),suggesting that glutamine deprivation had no significant effect on the stability of S100A4 mRNA.(2)The expression level of S100A4 in the Wnt/β-catenin pathway activator Ch IR-99021 treatment group was significantly higher than that in the control group under glutamine deprivation(P < 0.05),suggesting that glutamine deprivation down-regulates the expression of S100A4 by inhibiting the activity of Wnt/β-catenin pathway.Conclusion:1.Glutamine deprivation down-regulates S100A4 expression in MGC803 cells and HGC27 cells in a time-dependent and concentration-dependent manner.2.Glutamine deprivation inhibited the proliferation and migration of MGC803 and HGC27 cells in gastric cancer,and the cells were blocked in G2 phase.3.Transfection of S100A4 expression vector can partially recover the effects of glutamine deprivation on proliferation,migration and cell cycle arrest of MGC803 and HGC27 gastric cancer cells.4.Glutamine deprivation reduces the expression level of S100A4 by inhibiting the activity of Wnt/β-catenin pathway in gastric cancer cells MGC803. |
