A Preliminary Study On The Role And Mechanism Of Glutaminase In The Development Of Esophageal Squamous Cell Carcinoma

Background and PurposeEsophageal cancer has a higher morbidity and mortality rate in malignant tumors worldwide.According to histological type,it can be divided into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).In China,esophageal squamous cell carcinoma is the mainstay,in the pathogenesis of esophageal squamous cell carcinoma,abnormal energy metabolism of tumor cells is also one of the important factors.Glutamine(Gln)is a kind of non-essential amino acid rich in human cells,which is involved in biomacromolecules synthesis and energy metabolism.Gln not only participates in the growth and metabolism of normal cells,but also plays an important role in the development of tumor cells and provides necessary energy substances for the proliferation of tumor cells.In addition,Gln participates in the redox reaction of cancer cells to maintain cell activity,and glutamine metabolism is indispensable for the catalytic reaction of related enzymes.Glutaminase(glutaminase,GLS1)is the rate-limiting enzyme in the glutamine metabolic pathway,inhibiting the expression of GLS1,which can effectively inhibit the proliferation of cancer cells and even kill cancer cells.Glutamine energy metabolism plays an important role in many tumor cells,but it is not known how it regulates the occurrence and development of esophageal squamous cell carcinoma and what role it plays.Therefore,to clarify the role of GLS1 in esophageal squamous cell carcinoma and its mechanism for prevention and the treatment of esophageal squamous cell carcinoma has important scientific value and clinical significance.In this experiment,by analyzing the relationship between GLS1 expression and clinical parameters in esophageal squamous cell carcinoma tissues and adjacent tissues;and observing the effect of down-regulating GLS1 on the biological behavior of esophageal squamous cell carcinoma cells and β-catenin/p-GSK-3β/c-myc key protein expression in Wnt signaling pathway,in order to explore the impact of GLS1 on the development and mechanism of esophageal squamous cell carcinoma and provide a theoretical basis for GLS1 as a therapeutic target.Methods1.The immunohistochemical method was used to detect the expression of GLS1 protein in 70 esophageal squamous cell carcinoma tissues and 44 adjacent tissues and analyze the relationship between clinical parameters.2.Glutamine deficiency test to detect glutamine dependence of 6 differently differentiated esophageal squamous carcinoma cell.Normal esophageal epithelial cells HET-1A was used as a control.Filter strong cell.3.Western blot was used to detect the expression of-GLS1 protein in 6 esophageal squamous carcinoma cells with different degrees of differentiation.Using normal esophageal mucosal epithelial cells HET-1 A as a control,cell with higher GLS1 expression were screened.4.Clone formation experiment to detect the growth of cells in the absence of glutamine;siRNA transiention was used to detect the protein expression of GLS1 in two cells by western blot;The spectrophotometer was used to detect the changes in glutaminase activity in the two cells.5.CCK-8 test to detect cell proliferation;Scratch and transwell test to observe cell migration and invasion ability;Flow cytometry to detect cell cycle and apoptosis6.Flow cytometry detection of ROS changes in cells.7.Western blot detection of key β-catenin/p-GSK-3β/c-myc protein expression in Wnt signaling pathway in cells.Results1.Expression of GLS1 in esophageal squamous cell carcinoma and its relationship with clinical parametersThe expression of GLS1 protein in esophageal squamous cell carcinoma tissue is more in the cytoplasm,and there is a small amount of expression in the cell membrane and nucleus;Compared with adjacent tissues,the positive expression rate of GLS1 in esophageal squamous cell carcinoma tissue is higher(p<0.05);GLS1 protein expression is correlated with the pathological grade of clinical parameters in patients with esophageal squamous cell carcinoma.2.The results of glutamine deficiency experimentsIt can be seen that the lack of glutamine has little effect HET-1A and esophageal squamous cell carcinoma cells EC 109 and EC9706.Although the growth and proliferation of esophageal squamous carcinoma cells EC-1 and TE-1 were inhibited,the sensitivity to glutamine was not strong;But for esophageal squamous cell carcinoma TE13 and KYSE70 cells on days 1,3,5,and 7 high sensitivity to glutamine,indicating that TE13 and KYSE70 cells have a strong dependence on glutamine.3.Western blot detection of GLS1 protein expression in 6 kinds of esophageal squamous carcinoma cellsUsing HET-1A as a control,the expression of GLS1 protein in esophageal squamous cell carcinoma cell lines EC-1,EC 109,and EC9706 is low(p>0.05);Although the expression of TE-1 cell is high,it is not sensitive to glutamine in glutamine-sensitive screening experiments-The high expression of GLS1 protein in esophageal squamous cell carcinoma TE13 and KYSE70(p<0.01)and strong sensitivity to glutamine.4.Results of glutamine-deficient clone formation experimentsUsing gln-containing medium as a control,TE13 and KYSE70 cells grew well in Gln-containing medium and were able to aggregate into cell clusters,but in the absence of gln medium,the cell cluster formation was significantly inhibited and cell clone formation number decreased(p<-0.05).5 The effect down-regulating GLS1 on the activity of glutaminase in TE13 and KYSE70 cellsTaking siNC as a control,after transfection of siGLS1#1 and siGLS1#2,the activity of GLS1 in esophageal squamous cell carcinoma TE13 and KYSE70 cells decreased significantly.6.The effect of down-regulating GLS1 on the proliferation of TE13 and KYSE70 cellsTaking siNC as a control,siGLS1#1 and siGLS1#2 was significantly decreased at 24h,48h and 72h.It can be seen that down-regulating GLS1 can reduce the proliferation of esophageal squamous cell carcinoma ability.7.The effect of down-regulating GLS1 on the migration and invasion ability of TE13 and KYSE70 cells1)Migration capacity experiment:using siNC as a control,the migration rates of TE13 and KYSE70 cells transfected with siGLS1#1 and siGLS1#2 for 48 hours were significantly reduced(p<0.01).It can be seen that down-regulating GLS1 can reduce the migration capacity of esophageal squamous carcinoma cells.2)Invasion ability experiment:with siNC as the control,the invasive ability of TE13 and KYSE70 cells transfected with siGLS1#1 and siGLS1#2 for 48 hours was significantly reduced(p<0.01),which shows that down-regulating GLS1 can reduce the invasive ability of esophageal squamous cell carcinoma.8.The effects of down-regulation of GLS1 on TE13 and KYSE70 cell cycle and apoptosis1)Taking siNC as a control,the distribution of esophageal squamous carcinoma cell cycle in G2/M phase increased after 72h of siGLS1#1 and siGLS1#2 treatment(p<0.01),indicating that down-regulation of GLS1 blocked the cycle of esophageal squamous cell carcinoma in G2/M.2)With siNC as the control,the total apoptosis rates of esophageal squamous cell carcinoma TE13 and KYSE70 in siGLS1#1 and siGLS1#2 were increased after 72h of treatment(p<0.05).indicating that down-regulation of GLS1 can induce the induction of apoptosis.9.The effect of down-regulating GLS1 on ROS in TE13 and KYSE70 cellsTaking siNC as a control,the ROS level in cells of siGLS1#1 and siGLS1#2 groups increased significantly after treatment for 72h,indicating that down-regulation of GLS1 can increase the reactive oxygen species in the cells.10.The effect of down-regulating GLS1 on key protein in Wnt signaling pathway in TE13 and KYSE70 cellsTE13 and KYSE70 cells are transfected in the β-catenin/p-GSK-3β/c-myc signaling pathway the expression levels of β-catenin(p<0.05),p-GSK-3β,and c-myc decreased.ConclusionGLS1 is highly expressed in tissues and cells of esophageal squamous cell carcinoma;Down-regulation of GLS1 can reduce the proliferation,migration and invasion ability of esophageal squamous cell carcinoma,reduce glutaminase activity,block cell cycle,and increases apoptosis;The mechanism may be related to the increase of ROS level,the inhibition of key proteins in Wnt signaling pathway.