The Mechanism Of Sirt3 Regulating TIGAR Affecting The Sensitivity Of Non-small Cell Lung Cancer To Cisplatin

Background:Clinically,there are different treatment schemes for non-small cell lung cancer(NSCLC)in different stages.For a long time,the first choice for patients with stage Ⅰ、Ⅱ cancer is surgery and chemotherapy,but for patients with stage Ⅲ and Ⅳ metastatic lesions,this method has a poor prognosis.Nowadays,molecular detection of mutations in epithelial growth factor receptor(EGFR)and mesenchymal epithelial transforming factor(MET),as well as analysis of anaplastic lymphoma kinase(ALK)and proto-oncogene ROS1 translocation have become new methods for diagnosis of NSCLC,so targeted therapy can prolong the overall survival of NSCLC patients.However,most of the above treatment schemes require the adjuvant treatment of cisplatin,an anticancer drug.With the increase of the time taken by patients,the sensitivity of most patients to cisplatin gradually decreases or even reaches the level of drug resistance,leading to the drug treatment entering the platform stage,and the final treatment effect is poor.At present,people still lack of in-depth research on this phenomenon.A large number of studies have reported that cancer cells,even in an environment with sufficient oxygen,mainly rely on anaerobic glycolysis to obtain energy for themselves,that is,Warburg effect.NSCLC,which has reached the stage of cisplatin resistance,relies on this metabolic mode to provide energy for itself quickly.Therefore,we speculate that the change of metabolic mode may be one of the important reasons that affect the sensitivity of non-small cell lung cancer cells to cisplatin.Our previous study found that the expression of NAD-dependent mitochondrial deacetylase Sirtuin3(Sirt3)was higher in the immunohistochemical staining sections of patients with non-small cell lung cancer compared with normal lung tissue sections.Sirt3 can deacetyl ate various mitochondrial proteins by performing its deacetylase function,thus participating in the metabolic process of tumor cells.TIGAR(Tp53-induced glycolysis and apoptosis regulator),as a regulator of p53 induced glycolysis and apoptosis,is similar to the phosphofructose kinase 2/fructose-2,6-diphosphatase(PFK2/FBPase-2)in glycolysis.It inhibits the activity of PFK-1 by reducing the level of F-2,6-BP,reduces the glycolytic flux downstream of this point,and affects cell metabolism.Recent studies have shown that p53 independent TIGAR expression has been found in some human cancer cell lines,and these TIGAR still have the function of glycolytic enzyme.Therefore,exploring the relationship between Sirt3,TIGAR and aerobic glycolysis may provide a new way to change the sensitivity of non-small cell lung cancer to anticancer drug cisplatin.Objective:By studying the changes of glucose metabolism and drμg sensitivity of NSCLC under the action of Cisplatin,we explore the effect of Sirt3 on the Cisplatin sensitivity of NSCLC and the changes of glucose metabolism after the combined treatment,so as to provide new mechanism exploration and potential target for overcoming Cisplatin tolerance in clinical treatment of non-small cell lung cancer.Methods:1.Cispaltin with gradient concentration was applied to two kinds of non-small cell lung cancer cell lines NCI-H1299 and A549,and the effect of Cispaltin concentration on the viability was detected by MTT assay;Western blot was used to detect the expression of Sirt3 after Cisplatin treatment.2.Knocked down the expression of Sirt3 in NCI-H1299 and A549 cells,and combinated with low concentration of Cisplatin.The effect of Sh-Sirt3 and Cisplatin on the viability was detected by MTT assay;The effect of Sh-Sirt3 combined with Cisplatin on the apoptosis rate was detected by Annexin V-FITC/PI double staining and Western blot.3.Knocked down the expression of Sirt3 in NCI-H1299 and A549 cells and then combining with Cisplatin,the glucose uptake and lactate production of cells were detected with glucose and lactate kits;Western blot was used to detect the protein expression of glucose metabolism-related enzymes.4.Knocked down the expression of Sirt3 in NCI-H1299 and A549 cells,and then combining with Cisplatin,the specific location of Sirt3 and TIGAR in cells was checked by immunofluorescence assay and Western blot.5.Knocked down the expression of Sirt3 in NCI-H1299 and A549 cells,and then combining with Cisplatin,the effect of Sirt3 deacetylase function on the expression of TIGAR was detected by immunoprecipitation technique.6.Over-expressed TIGAR in NCI-H1299 and A549 cells.Western blot was used to reverse verify the effect of TIGAR expression change on Sirt3.7.The mechanism of Sh-Sirt3 enhancing the sensitivity of NSCLC to Cisplatin was verified based on the nude mice transplanted tumor model experiment.Results:1.Cisplatin inhibits the survival of NCI-H1299 and A549 cells and increases the level of Sirt3 protein.2.Compared with the control group,the survival rate of NCI-H1299 and A549 cells decreased after Sh-Sirt3.Compared with Cisplatin,the survival rate significantly decreased and the apoptosis rate significantly increased after Sh-Sirt3 combined with Cisplatin.Sh-Sirt3 enhanced the sensitivity of cells to Cisplatin.3.Sh-Sirt3 combined with Cisplatin in NCI-H1299 and A549 cells,the glucose uptake and lactic acid production in cells decreased.4.After NCI-H1299 and A549 cells Sh-Sirt3 combined with Cisplatin,compared with Conrol group,the expression of glycolytic enzymes TIGAR and PFK-1 decreased,and the expression of FBPasel increased.The expression of G-6-PD in NCI-H1299 cells did not change significantly,while the expression of G-6-PD in A549 cells decreased.5.Sirt3 and TIGAR are expressed and bound in the cytoplasm of NCI-H1299 and A549 cells.6.After Sh-Sirt3,the expression of acetylation protein and TIGAR acetylation increased in the cells,and Sirt3 played the role of deacetylase to regulate TIGAR.7.Compared with the control group,Sh-Sirt3 combined with Cisplatin significantly inhibited the growth of tumor in the nude mice model of NCI-H1299 non-small cell lung cancer cells.Conclusion:1.Sh-Sirt3 enhances the sensitivity of non-small cell lung cancer cells to Cisplatin and induces cell apoptosis.2.Sh-Sirt3 regulates TIGAR to inhibit NSCLC glycolysis and enhance cell sensitivity to Cisplatin.The mechanism is that Sh-Sirt3 can directly or indirectly promote TIGAR acetylation,increasing TIGAR expression level,further inhibiting the activity of enzyme PFK-1,reversing the cellular glycolytic process,and thereby enhancing sensitivity to Cisplatin.