Research On The Effect Of Fosmidomycin Inhibiting Neuron Specific Enolase In Gliomas

Objective:This research is aim to explore the effect and mechanism of fosmidomycin,the structural analogue of enolase inhibitor on tumor cell proliferation,so as to provide a new clue for antitumor drug research.Methods:From 2017 to 2018,18 glioma samples were collected from glioma patients who underwent pathological examination in the Pathology Department of the First Affiliated Hospital of Wannan Medical College.Immunohistochemistry was used to detect the expression of neuron specific enolase and calculate the percentage of positive area.According to malignant degree of gliomas,non-parametric analysis was performed on the expression levels of neuron-specific enolase in different degrees.U87 cell was cultured in experiment,and the expression of?-enolase was silenced by siRNA methods.Silence efficiency was tested by quantitative PCR and Western blotting experiment.Fosmidomycin was added in different concentration and different time after the best siRNA fragment was identified.Cell proliferation toxicity test was used to detect the proliferation toxicity of fosmidomycin added to U87 cells culture medium.Apoptosis experiment was used to determine whether fosmidomycin could cause apoptosis of U87 cells,then data was analyzed by multivariate analysis of variance.In order to explore the mechanism between fosmidomycin and neuron specific enolase,colorimetric method was used to detect the content of phosphoenolpyruvate in U87 cells.Then variance analysis was used.Results:1.The results of immunohistochemistry showed that the higher the malignant degree of gliomas was,the larger the percentage of positive area of immunohistochemical staining was,the higher the expression of neuron-specific enolase in glioma tissue(?~2=9.843,P=0.014).Further analysis showed that the expression of neuron specific enolase and the differences between different genders(H=-1.910,P=0.028)and tissue types(H=8.200,P=0.042)were statistically significant.The expression of neuron specific enolase in glioblastoma was higher than other types(?~2=8.200,P=0.042),and the expression of neuron specific enolase in male was higher than that in female(Z=-1.910,P=0.028).2.The small RNA interference experiment showed that the interference efficiency of the si-ENO1 B fragment was better than that of the si-ENO1 A fragment and the si-ENO1 C fragment.Three small RNA interference fragments were analyzed by quantitative PCR,and results indicated that the interference efficiency of si-ENO1B fragment was the most efficient(F=35.367,P<0.001).Western blot experiments were performed after cell protein extraction.Gray level ratios of the target protein(ENO1)and internal reference protein(GAPDH)under different siRNA interference were compared.The results were accordant with the quantitative PCR:The expression of?-enolase of si-ENO1 B fragment was lower than that of si-ENO1 A fragment and si-ENO1 C fragment(F=3.168,P=0.036).3.The results showed that different siRNA groups(F=91.255,P<0.001),different treatment times(F=233.226,P<0.001)and different concentrations of fosmidomycin(F=8.313,P<0.001)had different cytotoxicity on cell proliferation.The proliferation rate of the cells was also different in different siRNA groups(F=9.152,P=0.003).Under different treatment time,cell proliferation of different siRNA groups in different concentrations of fosmidomycin was also different(F=3.768,P=0.012).The higher the concentration of fosmidomycin was,the more toxic it was to U87 cells.4.Annexin V and PI double staining were used to detect the degree of apoptosis in the experimental group and the control group at different concentrations and at different times.The results showed that different siRNA groups(F=20.883,P<0.001)and fosmidomycin concentration(F=4.145,P=0.010)caused different apoptosis of U87 cells.Under different treatment time,the apoptosis rate was different in different si RNA groups(F=10.248,P=0.002)and different concentrations of fosmidomycin(F=5.639,P=0.002).Under different treatment time,different siRNA groups also had different apoptosis under different concentrations of fosmidomycin(F=3.528,P=0.021).The higher the concentration of fosmidomycin was,the more toxic it was to U87 cells.The results also showed that there was a difference in the content of PEP in cells under different treatment time(F=10.397,P=0.003).Conclusions:1.There was correlation between malignant degree of gliomas and the neuron specific enolase level.The higher the malignant degree was,the higher the neuron specific enolase content existed.2.Fosmidomycin may further interfere with the glycolysis pathway of malignant glioma cells through its interaction with neuron specific enolase,thus having a certain influence on the proliferation and growth of cells,but the degree of influence is limited.