Investigation On The Effect And Mechanism Of Tumor Suppressor NDRG2 Cooperating With The MTOR Inhibitor To Suppress The Aerobic Glycolysis And Cell Proliferation In Renal Cell Carcinoma

【Background】Renal cell carcinoma(RCC)is a kind of malignant tumor with high incidence,and it has been increasing every year.In recent years,the molecular targeted drug treatment of RCC has made great progress.However,the molecular targeted therapy of RCC still faces some problems: first-line TKIs drug treatment is easy to produce drug resistance,second-line mTOR inhibitor everolimus which has theoretically good effect,only has little effect on the clinical treatment of RCC.Some studies have shown that RCC is a kind of metabolic disease,and aerobic glycolysis provides an important material and energy source for its malignant proliferation.It has also been reported that mammalian rapamycin target protein(mTOR)is closely related to aerobic glycolysis.Our previous studies showed that N-myc down stream regulated gene 2(NDRG2)can also regulate aerobic glycolysis.In this study,we tried to explore the effect of mTOR inhibitors on the aerobic glycolysis of RCC,and the effect of mTOR inhibitors on the aerobic glycolysis and malignant proliferation of RCC after overexpression of NDRG2 gene.【Objectives】1.The activation of mTOR signaling pathway in RCC.2.The effect of mTOR inhibitor on aerobic glycolysis and cell proliferation of RCC3.The effect of everolimus on aerobic glycolysis-related enzymes in RCC4.The effect of overexpression of NDRG2 on the activity of mTOR signaling pathway5.The synergistic effect of everolimus and NDRG2 on aerobic glycolysis and cell proliferation of RCC【Methods】1.The expression and phosphorylation of mTOR singal pathway in RCC and its adjacent tissues were detected by Western blot and immunohistochemical staining.2.The glucose consumption and lactate production of RCC cells treated with mTOR inhibitors were detected by a glucose assay kit and lactate assay kit;the acid production rate of renal carcinoma cells treated with mTOR inhibitors were detected by a Seahorse Bioscience XFe24 Extracellular Flux Analyzer;the cell viability of RCC cells treated with of mTOR inhibitors were detected by cell counting kit-8(CCK-8).3.Immunoblotting was used to detect the expression of aerobic glycolysis-related enzymes and HIF1α protein in RCC treated with everolimus,and real-time quantitative PCR was used to detect the expression of HIF1α m RNA in RCC cells treated with everolimus at different time points.4.Infection with lentivirus containing NDRG2 was used to overexpress NDRG2 in RCC cells,and then western blot was used to detect the expression and phosphorylation of mTOR and its downstream molecules.5.Western blotting was used to detect the expression of aerobic glycolysis-related enzymes and HIF1α in RCC cells which were overexpressed NDRG2 or m Cherry and treated treatment with everolimus,the glucose consumption and lactate production of RCC cells which were overexpressed NDRG2 or m Cherry and treated treatment with everolimus were dected by a glucose assay kit and lactate assay kit,and CCK-8 was used to detect the cell viability of RCC cells which were overexpressed NDRG2 or m Cherry and treated treatment with everolimus.The malignant proliferation of RCC tumor tissue in the overexpressed NDRG2 gene and its control group was detected by everolimus gavage in nude mice.【Results】1.The phosphorylation level of mTOR signaling pathway related molecules P70S6K(Ser371)and 4EBP1(Thr37 / 46)in RCC tissue was higher than that in paracancerous tissue,and the activation level of phosphorylated 4EBP1(Thr37 / 46)was significantly correlated with RCC pathological grade(P < 0.0001),and the higher the pathological grade,the higher the activation level of phosphorylated 4EBP1(Thr37 / 46).2.mTOR inhibitors can inhibit the glucose consumption,lactate production and proliferation of RCC cells.3.The expression of HIF1α and aerobic glycolysis-related enzymes in RCC cells was decreased with the increase of treatment time of everolimus.When cobalt chloride was used to block the degradation of HIF1α,it was found that the inhibitory effect of everolimus on the expression of aerobic glycolysis related enzymes,glucose consumption and lactate production of RCC cells could also be reversed.4.The activation of mTOR signaling pathway and the expression of HIF1α in the NDRG2overexpressed-cells were significantly downregulated than that in the control group(P< 0.05).5.The expression of HIF1α and aerobic glycolysis-related enzymes,as well as glucose consumption and lactate production in the everolimus treatment overexpressed NDRG2 group were significantly downregulated than that in the control group(P < 0.01).The growth in everolimus treated RCC tumor tissue after over expression of NDRG2 gene was significantly slower than that in the control group(P < 0.01),and the expression of Ki67 in everolimus treated RCC tumor tissue after over expression of NDRG2 gene was lower than that in the control group.【Conclusions】1.mTOR signaling pathway is highly activated in renal cell carcinoma,2.mTOR inhibitor can inhibit aerobic glycolysis and cell proliferation of RCC cells,3.Everolimus can inhibit aerobic glycolysis of renal cancer cells by inhibiting HIF1α,4.NDRG2 can inhibit the activation of mTOR signal pathway in RCC cells,5.NDRG2 cooperates with the mTOR inhibitor to suppress the aerobic glycolysis and cell proliferation in renal cell carcinoma.