The Role Of Glycolysis In Bladder Tumor Immune Escape Mechanism

Objective: To study the relationship between the key enzyme levels HIF-1? and PFKFB3 and the immune checkpoint PD-L1 in the glycolysis pathway of bladder tumor cells at the cell level,and to construct a bladder tumor in situ model to easily and rigorously prove the sugar in bladder tumor cells.there is a certain relationship between glycolysis and the immune escape mechanism of bladder tumor cells.And further exploring its possible effects on the proliferation and migration of bladder tumor cells,thus finding a new direction for the treatment of bladder tumors.Methods: Using bioluminescence technology,construct a plasmid vector including green luciferase gene and GFP fluorescent protein gene.Firstly,the plasmid was transformed and extracted to obtain a large number of plasmids.After preparing the lentivirus,the lentivirus was concentrated,transfected by the lentivirus,and cultured first Mouse Mb49 bladder tumor cells(Mb49-GFLU)carrying the luciferase gene used luciferase as a carrier to detect the luminescence of the tumor tissue.At the cellular level,the glycolysis pathway was affected by the addition of inhibitors of HIF-1? and PFKFB3 in the glycolysis pathway,and the HIF-1? and PFKFB3 inhibitor YC-1 and PKF015 were added to the glycolysis pathway by Western Blot and RT-qPCR methods.The glycolysis pathway was changed and the expression of PD-L1 in bladder tumor cells was detected to verify the relationship between the bladder tumor immune escape mechanism and glycolysis.Then from the animal level,Balb / c mice were used,after injecting Mb49-GFLU into the bladder mucosa of mice,an in situ model of mouse bladder tumors was constructed.After tumor formation,regular inhibitors of HIF-1? and PFKFB3 were injected intraperitoneally.According to the animal's fluorescence expression,it was easy to find whether the tumor was inhibited and immunized histochemical confirmation of tumor tissue,and further verification by Western Blot method was carried out.Results: After transfection of Mb49 cells with the lentiviral vector carrying the green luciferase gene,the expression of green fluorescent protein can be seen under a fluorescence microscope,and it has been confirmed that the transfection has been successful after detection by luciferase.After normal cultivation of Mb49-GFLU cells,and after setting the control group,HIF-1? and PFKFB3 inhibitors YC-1 and PFK015 and twoinhibitors were added respectively.Western Blot and RT-qPCR methods were used to detect the level of HIF-1? and PFKFB3 and tumor expression PD-L1.It was found that compared with the control group,the expression levels of HIF-1? and PFKFB3 in the three experimental groups decreased,the glycolysis level decreased,the tumor expression level decreased,and the tumor immune escape mechanism was inhibited.The mouse bladder in situ model was constructed by aseptic surgery.Mb49-GFLU cells expressing fluorescent genes were injected into mouse bladder mucosa.One week later,the tumors were observed under the animal imaging instrument;the control group,the HIF-1? inhibitor YC-1 group,and the PFKFB3 inhibitor PFK015 group were added to the control group.Both inhibitors were added to the Balb/c mice of the group,and the bladder was transfected with tumor cells in situ.After different treatments for 2 weeks,the tumor tissues were extracted and tested by Western Blot method,and we checked the tumor tissue according to immunohistochemistry.It can be found that after the addition of inhibitors,HIF-1? and PFKFB3 in the glycolysis pathway are reduced,and the expression of PD-L1 in the tumor tissue is also reduced.The results of immunohistochemistry are consistent with the experimental results which thus verifying the above results.Conclusion: This study found a new way to directly verify the expression of bladder tumors through the mouse bladder tumor in situ model.It has been shown that the glycolysis level of bladder tumor cells is positively correlated with tumor PD-L1 expression level at the cellular and animal levels.When the solution level is suppressed,the tumor PD-L1 expression level also decreases,and the immune escape mechanism is suppressed.Therefore,the emerging treatment of bladder tumors can be studied through the molecular mechanism of HIF-1? and PFKFB3.