Regulation Of Glycolysis And Inhibition Of CD4~+/CD8~+T Cell Immunity In Esophageal Tumorigenic Tissue Of Mice By PLCE1/ENO1

Objective: To investigate the glycolysis level and CD4+/CD8+T cell immune function in C57BL/6 PLCE1 mice with different genotypes treated with 4NQO drug and ENO1 inhibitor drug.Methods:(1)Esophageal cancer model was constructed in mice induced by 4MQO drinking method.(2)Glucose,lactic acid,pyruvate and ATP test kit were used to detect glycolysis product levels.(3)Bioinformatics predicted the correlation between ENO1 expression level and T cells.(4)The relationship between PLCE1 and ENO1 expression levels and T cell infiltration levels in esophageal squamous cell carcinoma was detected by multicolor fluorescence technique.(5)m RNA levels of IFN-γ,TNF-α,Granzyme B and PD-1 were detected by q PCR.(6)The proportions of CD45+T cells,CD3+T cells,CD4+T cells and CD8+T cells in gene mouse esophageal carcinoma were determined by flow cytometry.(7)Flow cytometry was used to detect the proportion of CD8+T cells expressing IFN-γ,TNF-α,Granzyme B and PD-1.(8)Flow cytometry was used to detect the proportion of CD4+T cells expressing IFN-γ,TNF-αand PD-1.Results:(1)Compared with the wild-type,homozygous and homozygous groups,the glucose level of the wild-type control group was decreased(2.128±0.158vs2.653±0.162vs2.529±0.286vs2.972±0.270,P<0.001).Lactic acid level increased(3.260±0.598vs2.103±0.753vs2.080±0.252vs1.555±0.718,P<0.001),ATP levels increased(217.164±60.561vs105.887±83.626vs100.505±52.988,P=0.016),Pyruvate levels increased(0.048±0.001vs0.026±0.006vs0.024±0.006vs0.012±0.003,P=0.005),Similarly,the m RNA level of hexokinase increased(1.677±0.097vs1.203±0.166vs1.001±0.060vs0.419±0.050,P<0.001),the m RNA level of pyruvate kinase increased,However,m RNA levels of pyruvate kinase were statistically significant only between the wild-type control group and the homozygous treatment group(0.556±0.031vs0.503±0.035vs0.558±0.034vs0.457±0.026,P=0.0002).The m RNA levels of phosphofructokinase were statistically significant(0.734±0.063 vs 0.465±0.192vs0.674±0.254vs0.463±0.054,P=0.0351).(2)When ENO1 expression level was high,the infiltration level of CD4+/CD8+T cells decreased(P<0.05).(3)In the wild-type control group,CD45+T cells(13.867±1.703vs18.277±4.681vs17.505±3.392vs23.823±2.921,P<0.01),CD3+T cells(13.867±1.703vs18.277±4.681vs17.505±3.392vs23.823±2.921,P<0.001),CD4+T cells(13.867±1.703vs18.277±4.681vs17.505±3.392vs23.823±2.921,P<0.001)and CD8+T cells(13.867±1.703vs18.277±4.681vs17.505±3.392vs23.823±2.921,P<0.001)were significantly lower than those in wild-type treatment group,homozygous control group and homozygous inhibitor group.(4)The m RNA level of FN-γ in tumor tissue in wild-type control group was significantly lower than that in wild-type treatment group,homozygous control group and homozygous inhibitor group(5.836±0.382vs8.717±0.824vs7.770±0.438,P<0.001).On the contrary,the expression level of PD-1 was increased(0.480±0.019vs0.389±0.065vs0.370±0.052vs0.267±0.051,P<0.001).In the wild-type control group,TNF-α(1.608±0.162vs2.135±0.645vs2.310±0.414vs2.529±0.106)in tumor tissue,P=0.013)and Granzyme B(18.79±2.255 vs 19.85±1.864vs18.92±2.014vs22.27±1.037,P=0.031)m RNA levels were significantly lower than those in homozygous treatment group.There was no statistical significance compared with wild-type treatment group and homozygous control group.(5)FN-γ of CD8+T cells in tumor tissue in homozygous inhibitor group(4.48±0.622vs7.74±2.482vs10.007±2.541vs18.897±6.052,P<0.001),TNF-α(0.657±0.048vs1.007±0.116vs0.953±0.311vs1.535±0.167,P<0.001)and Granzyme B(13.867±1.703vs18.277±4.681vs17.505±3.392vs23.823±2.921,P<0.001)were significantly higher than those in wildtype treatment group,homozygous control group and homozygous inhibitor group.On the contrary,the expression level of PD-1(1.36±0.167 vs 0.933±0.156vs0.808±0.134vs0.545±0.077,P<0.001)decreased.(6)FN-γ of CD4+T cells in tumor tissue in homozygous inhibitor group(8.623±3.388vs12.633±2.186vs16.683±3.473vs23.433±4.244,P<0.001)and TNF-α(0.653±0.040vs0.787±0.080vs0.918±0.063vs1.29±0.289,P<0.001)were significantly higher than those in wild-type treatment group,homozygous control group and homozygous inhibitor group.On the contrary,the expression level of PD-1(9.073±1.59vs5.887±1.153vs5.797±1.456vs3.79 ± 0.682,P<0.001)increased.