| Objective:The difference of serum metabolites between patients with esophageal squamous cell carcinoma and normal subjects was analyzed by untargeted metabolomics.To study the effect of PLCE1 on lactic acid secretion in esophageal squamous cell carcinoma through ENO1,and explore the effect of lactic acid on intracellular pH,apoptosis,ATP synthesis,cytokine secretion and PD-1 expression of CD8~+T cells.Methods:(1)Serum samples from 7 patients with esophageal squamous cell carcinoma and 7 normal subjects were collected and divided into esophageal squamous cell carcinoma group and normal control group.Non-targeted metabolomics based on LC-MS/MS technology was used to evaluate the qualitative quantitative and data quality of serum metabolites.The structure of serum metabolites was identified and rated according to the reliability level of metabolite identification;(2)The number of serum metabolites identified after the combination of positive and negative ions was counted and chemical classification and classification statistics were carried out.Univariate statistical analysis,PLS-DA model and OPLS-DA model were used to analyze whether there were differences in serum metabolites between the esophageal squamous cell carcinoma group and the normal control group;(3)The effects of PLCE1 and ENO1 on lactic acid secretion and glucose utilization were determined by detecting lactic acid and glucose level in the supernatant of esophageal squamous cell culture;(4)Human peripheral blood CD8~+T cells were selected by magnetic bead sorting and the positive rate of CD8~+T cells was determined by flow cytometry(FCM);(5)The effects of lactic acid on intracellular pH of CD8~+T cells,apoptosis of CD8~+T cells and ATP synthesis were detected by FCM;qRT-PCR and FCM were used to detect the effect of lactic acid on the secretion of IL-2 and IFN-γand the expression of PD-1 by CD8~+T cells.Results:(1)A total of 7 serum samples from patients with esophageal squamous cell carcinoma and 7 normal human serum samples were included for testing.Principal component analysis of total samples,correlation of QC samples and Hotelling’s T2 test of total samples showed that the experiment had well repeatability,and multivariate control chart of QC samples and relative standard deviation reflected perfect instrument stability.The qualitative and quantitative data of this experiment can be used for subsequent analysis.According to the reliability level of metabolite identification defined at the Metabolomics Society Annual Meeting in Brisbane,Australia in 2017,the level of metabolite identification in this experiment was level 2.(2)A total of 266 metabolites were identified by positive and negative ion modes,and 164 and 102 were identified by the two modes respectively.Among the metabolites classified by chemical classification,lipids and lipid-like molecules were the most(49 in total,accounting for 18.421%),such as 16-hydroxypalmitic acid,acetylcarnitine,α-linolenic acid,eicosapentaenoic acid(EPA),2-hydroxy-3-methylbutyric acid,anoxycholate,etc.Organic acids and derivatives(42 kinds,accounting for 15.789%),such as lactic acid,1-aminocyclopropane carboxylic acid,3-methylhistidine,betaine,2-oxyadipic acid,ketoisocaproic acid,etc.Univariate and multidimensional statistical analysis showed that serum metabolites were different between the esophageal squamous cell carcinoma group and the normal control group;(3)After 36h of culture of esophageal squamous cell cells,the lactic acid level of culture supernatant in si-PLCE1 group(4.181±0.204)was significantly lower than that in control group(5.357±0.147)(P<0.001),the lactic acid level of supernatant in overexpressed ENO1 group(7.806±0.196)was significantly higher than that in control group(P<0.001),the level of glucose(4.339±0.083)in culture supernatant of PLCE1 group was significantly higher than that of control group(3.206±0.019),and the level of lactic acid(1.762±0.019)in culture supernatant of overexpressed ENO1 group was lower than that of control group(P<0.001);(4)Human peripheral blood CD8~+T cells were sorted by Mitineyi magnetic beads and cultured in vitro,the positive rate of CD8~+T cells was more than 90%by flow cytometry(FCM);(5)pH in CD8~+T cells decreased with the increase of lactic acid concentration in the medium;the apoptosis rate of CD8~+T cells in 20 mM LA group was(91.863±5.997)%,which was significantly higher than that in control group(P<0.001);in CD8~+T group stimulated by PMA/lono,ATP levels in 10 mM LA group(317.457±34.641)and 15 mM LA group(263.896±44.006)were lower than those in control group(375.457±51.020),there was significant difference between control group and 15 mM LA group(P<0.05).The secretion levels of IL-2 and IFN-γby CD8~+T cells were detected by FCM;the secretion levels of IL-2 and IFN-γwere decreased in both 10mM LA group and 15 mM LA group,and the decrease was more obvious in 15 mM LA group,but not in pH6.4 group.The inhibition of IL-2 and IFN-γin 15 mM Na L group was weaker than that in 10 mM LA group and 15 mM LA group.The expressions of IL-2 and IFN-γin CD8~+T cells of PMA/lono stimulation group were detected by qRT-PCR,and the expressions of IL-2 and IFN-γmRNA in LA group were significantly lower than those in control group(P<0.001).The expression of PD-1 in CD8~+T cells was detected by FCM,the positive rate of PD-1 in LA group was significantly lower than that in control group(100.000±0.947)(P<0.01),the mRNA level of PD-1 in the LA group(2.784±1.403)was significantly lower than that in the control group(15.304±3.492)by qRT-PCR(P<0.001).Conclusions:(1)There were differences in serum metabolites between esophageal squamous cell carcinoma patients and healthy subjects;(2)PLCE1 regulates ESCC lactic acid secretion through ENO1;lactic acid can reduce the intracellular pH of CD8~+T cells,promote apoptosis of CD8~+T cells,inhibit ATP synthesis,cytokine secretion and PD-1 expression of CD8~+T cells. |
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