The Anti-tumor Role And Mechanism Of SIRT3 In Gastric Cancer

Background:Gastric cancer(GC)is the fourth most common malignancy and the second leading cause of cancer-related death in the world.Currently,surgical resection,chemotherapy and radiotherapy remain to be the mainstay of gastric cancer treatment.Current research on the etiology and progression of gastric cancer mechanisms showed that in addition to genetic factors,many important molecular events are the underlying causes of tumor formation,development and metastasis.Although great progress has been achieved in the study of the gastric cancer in recent decades,the molecular mechanism of gastric cancer initiation and progression still remains elusive.Silencing information regulator 3(SIR.T3)is an important deacetylase in the mitochondria,and plays a critical role in coordinating multiple mitochondrial metabolism,and balance the stability of the internal environment.SIRT3 is highly expressed in heart,liver,kidney and skeletal muscle which have an active metabolism.Accumulating evidence has recently demonstrated that SIRT3 plays an important role in the process of tumor growth,reproduction,differentiation,senescence and apoptosis.However,the biological function of SIRT3 in gastric cancer has been poorly characterized,it is definitely worth further investigation.Objective:Compare the difference of SIRT3 expression between gastric cancer and normal tissues;Compare the expression of S1RT3 in gastric cancer cells and normal gastric epithelial cells;To analyze the correlation between the expression level of SIRT3 in patients with gastric cancer and the clinicopathological features;To analyze the effect of SIRT3 expression on the prognosis of gastric cancer patients;To investigate the ef'fect of SIRT3 on the proliferation,apoptosis and invasion of gastric cancer cells;Investigate the tumor suppressive function of SIRT3 in gastric cancer cells;Verify the potential target of SIRT3;To investigate the regulatory action of SIRT3 on Notch-1 signaling pathway,and explore possible molecular regulation mechanisms.Methods:1.Quantitative real-time PCR and Western blot were used to detect the expression of SIRT3 in the human gastric cancer cell lines AGS,SGC-7901 and BGC-823 and the normal gastric epithelium cell line GES;2.Collect specimens from patients with gastric cancer,Quantitative real-time PCR,Immunohistochemistry and Western blot were used to detect the expression of SIRT3 in gastric cancer tissues;3.The clinicopathological data of patients with gastric cancer were collected.The expression of SIRT3 was correlated with the clinical and pathological characteristics of patients by immunohistochemistry.To study the relationship between SIRT3 expression and prognosis of gastric cancer.4.SIRT3-siRNA was transfected into AGS cells by Iipofectamine to interfere with SIRT3 gene expression.The proliferations of gastric cancer cells were determined using a CCK-8 assay.Cell clone formation was assessed by colony formation assay.Investigate the effect of SIRT3 gene silencing on the proliferation of tumor cells.5.Construct the recombinant plasmid pcDNA3.1(+)-SIRT3,AGS Cells were transfected with lipofectamine 2000 according to the instruction.To investigate the effect of SIRT3 overexpression on the proliferation of tumor cells,the proliferations of gastric cancer cells were determined using a CCK-8 assay according to the manufacturer's protocol.Cell clone formation was assessed by colony formation assay.6.The effects of SIRT3 on the cell apoptosis,cell migration in Trans-well cells and the tumorigenic ability of AGS cells in nude mice were observed.7.Quantitative real-time PCR and Western blot were used to detect the expression of Notch-1 in each group cells.To observe the regulatory relationship between SIRT3 and Notch-1.8.SIRT3 and Notch-1 were co-transfected into AGS cells,the proliferation of tumor cells was observed using a CCK-8 assay and colony formation assay.Results:1.SIRT3 expression in several gastric cancer cell lines including AGS,SGC-7901 and BGC-823 cells were analyzed by qRT-PCR and western blot.We found that SIRT3 was dramatically decreased both at the mRNA and protein levels in three GC cell lines compared with normal gastric epithelium cell line GES.2.Data showed that mRNA expression of SIRT3 in GC tissues was significantly down-regulated compared with the normal tissues.Western blot analysis indicated that the SIRT3 protein expression was also reduced in GC tumor tissues.3.The expression level of SIRT3 in gastric carcinoma was correlated with the stage of tumor in patients with gastric cancer.The later the clinical stage,the lower the expression level of SIRT3.4.There was no significant correlation between the expression of SIRT3 and the prognosis of patients with gastric cancer.Multivariate analysis showed that the main factors influencing the prognosis of patients with gastric cancer were lymph node metastasis and tumor TNM staging.The more the lymph node metastasis,the worse the prognosis of gastric cancer patients,and tumor TNM staging later,the worse the prognosis of patients.5.SIRT3 inhibits the proliferation,migration and invasion of gastric cancer cell.To investigate the biological role of SIRT3 in gastric cancer,AGS cells were transfected with plasmids encoding SIRT3.qRT-PCR and western blot confirmed the upregulation of SIRT3 in AGS cells.The CCK-8 assay and colony formation assay showed that SIRT3 overexpression significantly inhibited the proliferation ability and colony formation numbers of AGS cells.In addition,we interfered with SIRT3 expression with small interfering RNA(siRNA)in AGS cells.Consequently,the growth curves showed that cell growth was obviously enhanced after SIRT3 knockdown in AGS cells.The colony formation assay displayed a dramatic increase in colony number when AGS cells were transfected with the SIRT3 siRNA.Overexpression of SIRT3 can promote AGS cell apoptosis,inhibit cell migration and invasion,and can inhibit AGS cells in nude mice subcutaneous tumor.Taken together,our results demonstrated that SIRT3 might be an growth suppressor in gastric cancer cells.6.SIRT3 inhibited Notch-1 expression in AGS cells.We furthermore investigated the molecular mechanism underlying the inhibitory effects of SIRT3 on gastric cancer cells.Surprisingly,over-expression of SIRT3 reduced the mRNA expression of Notch-1 in AGS cells.In addition,westerm blot analysis also showed that Notch-1 protein expression was significantly decreased in AGS cells overexpressing SIRT3.By contrast,down-regulation of SIRT3 with siRNA obviously elevated the Notch-1 expression both at mRNA and protein levels.These results showed that SIRT3 negatively regulated Notch-1 expression in AGS cells.7.Inhibitory effects of SIRT3 was mediated by up-regulation of Notch-1.In order to explore how SIRT3 exhibited an inhibitory effect on gastric cancer cells growth,the expression of Notch-1 was up-regulated after transfection with plasmids encoding Notch-1.As a result,the inhibitory effects of SIRT3 on cell proliferation and colony formation were partially reversed after overexpression of Notch-1 in AGS cells.Taken together,these data showed that SIRT3 could suppress AGS cell proliferation by down-regulation of Notch-1.Conclusions:In conclusion,our results demonstrated that SIRT3 expression in gastric cancer cells and tissues decreased.The expression level of SIRT3 in gastric carcinoma was correlated with the stage of tumor in patients with gastric cancer.The later the clinical stage,the lower the expression level of SIRT3.The expression level of SIRT3 was not associated with prognosis.Overexpression of SIRT3 can inhibit the proliferation,migration and invasion of gastric cancer cells and inhibit AGS cells from subcutaneous tumor formation in nude mice.SIRT3 and Notch-1 presence regulatory relationship.SIRT3 suppresses the proliferation of gastric cancer cells via inhibition of Notch-1 expression and might provide novel therapeutic targets in the gastric cancer treatment.