SIRT1 Gene Expression And The Research Of The Mechanism In Gastric Cancer Tissue

Objective:To investigate the expression of SIRT1 and ?-catenin in gastric cancer and explore its correlation with clinical pathological features. Clone SIRT1 full-length cDNA, construct containing SIRT1 gene and its mutant T200I, E420K recombinant eukaryotic expression vector and thus further study the biological function of SIRT1 gene and its mutants in the process of gastric carcinogenesis.Methods:Collected the 101 cases of gastric cancer tissue of surgical removal in binzhou medical university affiliated hospital, used immunohistochemical method to detect the expression of SIRT1 and P-catenin in gastric cancer tissue and analyzed its correlation with tumor clinical pathological features. RT-PCR amplified SIRT1 gene full-length cDNA. PCR products were cloned into the eukaryotic expression vector pcDNA3.1(+) through double digestion. We got pcDNA3.1(+)-SIRTl recombinant plasmid. While using site-directed mutagenesis to build its mutant pcDNA3.1(+)-T200I and pcDNA3.1(+)-E420K expression vector. Recombinant plasmid was identified by enzyme digestion and DNA sequencing and by western blotting confirmed recombinant eukaryotic expression vector to construct successfully from the protein level. We prepared competent cells, transformd plasmid and extracted plasmid to obtain plenty of pcDNA3.1 (+), pcDNA3.1 (+)-SIRT1, pcDNA3.1 (+)-T200I, pcDNA3.1 (+)-E420K eukaryotic expression vector, and then transfected it into 293T cells by using the method of liposome transfection. MTT and flow cytometry were used respectively to measure the cellular proliferation and cell cycle of five groups containing the blank control group of nothing DNA transfection (blank), empty carrier pcDNA3.1 (+) group (no load), the restructuring of carrier pcDNA3.1 (+)-STRT1 group (WT), pcDNA3.1 (+)-T200I (T200I group), pcDNA3.1 (+) E420K (E420K group). Western blotting test the cell cycle related proteins Cyclin Dl expression in five groups of cells. SIRT1 inhibitor NAM treated 293T cells and tested the cell proliferation in vitro and the expression of Cyclin D1.Results:The SIRT1 and ?-catenin positive expression rate were 64.4%(65/101) and 47.5%(48/101) in gastric cancer tissues. Statistically, the expression of SIRT1 in intestinal type gastric cancer were significantly higher than those in diffuse type gastric cancer (P<0.05). Altered expression of SIRT1 was not associated with clinicopathological parameters, including tumor location, size and lymph node metastasis(P>0.05). There was no significant correlation between SIRT1 and ?-catenin expression (r=0.01, P=0.751). SIRT1 gene was successfully cloned full-length cDNA, and successfully constructed pcDNA3.1(+)-SIRT1 eukaryotic expression vector and its mutant. Positive recombinant plasmid sequencing was compared after enzyme digestion, completely consistent with the expected sequence. Transfected 293T cells were established with His tagged SIRT1 protein expression by western blotting experiment. We successfully prepared competent cells, extracted plasmid and transfected of 293T cells. MTT experiments showed that the cell proliferation capacity of recombinant vector pcDNA3.1 (+)-STRT1 group (WT), pcDNA3.1 (+)-T200I (T200I group) and pcDNA3.1 (+)-E420K (E420K group) is higher than the blank control group of nothing DNA transfection (blank) and empty carrier pcDNA3.1 (+) group (no-load) (P<0.05). And the cell proliferation ability between three groups containing WT group, T200I group and E420K had no significant difference (P>0.05). Cell proliferation in vitro was reduced after SIRT1 inhibitor NAM treating 293T cells. The cell cycle analysised by flow cytometry, the results showed that the growth scores(S+G2 cell proportion) of WT group, T200I group and E420K three groups of cells is higher than that of blank group and no load cells (P<0.05), but the three group of cells in the cell cycle change was not significant (P>0.05). Western blotting experiments have confirmed that the cell cycle related proteins Cyclin D1 is overexpression in the WT group, the T200I group and E420K group of three groups of cells.With SIRT1 inhibitor treated cells Cyclin D1 lower expression.Conclusion:This study confirmed that SIRT1 expression was significantly higher in the intestinal type of gastric cancer than diffuse gastric cancer, illustrate that SIRT1 may play a different role in different histological types of gastric cancer.We successfully constructed the SIRT1 gene recombinant plasmid pcDNA3.1 (+)-SIRT1 and its mutant pcDNA3.1 (+)-T200I, pcDNA3.1 (+)-E420K eukaryotic expression vector, in order to provide enough genetic material for SIRT1 gene and its mutant T200I, E420K biological functional studies. The upregulation of SIRT1 and its mutant genes can promote cell proliferation in vitro and the progress of the cell cycle, and the downregulation of SIRT1 gene expression inhibit cell proliferation in vitro and the progress of the cell cycle, its mechanism may be related to change the expression of cell cycle related protein CyclinDl. This research shows that SIRT1 maybe play the role of promoting cancer gene in the occurrence and development of gastric cancer, as it provides the potential theory basis for using SIRT1 inhibitors in the clinic. While the SIRT1 mutant T200I and E420K did not change the effects of the SIRT1 gene on cell proliferation and cell cycle.