| Gastric cancer is one of the most common disease of malignant tumors in digestive system which are harmful to health.There are nearly900thousand new cases worldwide and about400thousand new cases in China.Compared to other countries, the morbidity and mortality of gastric cancer are higher in China. Surgical resection is the main method to treat this disease.With the development of chemotherapy,radiotherapy and immunotherapy,especially the using of MDT, the treatment of gastric cancer made a big progress,but the status of high mortality isn’t changed. The overall and disease free survival rate is still very low. With the development of molecular biology technology, many molecule and signal channel were confirmed to make impact on the malignant biological behavior of gastric cancer. But the five year survival rate of advanced gastric cancer is still only20-30%, even if lots of molecule-targeting agents being used.Therefore, it is urgently necessary to identify new molecule and signal channel involved in the mechanism of pathogenesis and development for gastric cancer, which will provide the theoretical base and experimental proof for the novel diagnosis and treatment for gastric cancer.Acetylation and deacetylation are the important ways ans methods in regulating the pathophysiological process of body. SIRT1(Silent information regulator1)SIRT1is an NAD+-dependent deacetylase essential for normal embryonic development, differentiation and homeostasis. The remarkably broad range of SIRT1functions is achieved by targeted deacetylation of key regulatory proteins involved in selective gene expression. SIRT1targets include transcription factors and cofactors involved in metabolic regulation.SIRT1plays an important role in health and disease.Interacting with histone and nonhistone such as P53and PGC-1α, SIRT1plays a important role in mammalian metabolism.The deacetylation effect of SIRT1on the histone and nonhistone can play a part in cells growth,apoptosis,also the occurrence and development of tumor.But the effect of SIRT1is different between different organs.Debates have continued about whether SIRT1is oncogene or anti-oncogene.The research of SIRT1in gastric cancer is rarely mentioned.We will explore the effect of SIRT1in gastric cancer through the research of SIRT1expression in tissues and cells of gastric cancer.STAT3is a multifunctional transcription factor,which can be activated by cytokines, growth factors and oncogenes.It could enhance proliferation, inhibit apoptosis, induce angiogenesis and invade immune surveillance. STAT3was therefore considered as an oncogene. STAT3is a cytokine-responsive transcription factor regulated by SIRT1deacetylation and known to suppress PGCl-α transcription. It is reported that STAT3expression is high in gastric cancer. Lots of evidences indicated that the abnormal activation of JAK/STAT is very important in the pathomechanism of gastric cancer.The activation of JAK/STAT is instantaneous in normal tissue but is constitutively in gastric cancer.The activation of JAK1/STAT3is proved to be important in the occurrence of gastric cancer.We will explore the relationship between SIRT1and STAT3by the research of co-expression in gastric cancer,finding the molecular mechanism in the progress of gastric cancer.We will also use the STAT3knock out transgenic mice to build gastric cancer model,and contrastively study the SIRT1expression.We will try to find the signal channel and the key point of target therapy for SIRT1in gastric cancer,providing the theory evidence for the treatment of gastric cancer.Objective:1, To detect the SIRT1expression in tissues and cells of gastric cancer.To study the relationship between SIRT1expression and clinical pathological characteristics for tumor.2, To study the effect of SIRT1in gastric cell proliferation and apoptosis.3, To explore the possible molecular mechanism and signal channel of SIRT1in progress of gastric cancer.Methods:1, We detected the SIRT1mRNA expression in fresh gastric cancer by real-time PCR analysis;2, We tested the SIRT1expression in gastric caner cells by using immunoblotting and RT-PCR in protein and mRNA level;3, We compared the SIRT1expression in gastric cancer tissue with the non-tumor mucosa by immunohistochemical staining and image analysis;4, We analyzed the relativity between the SIRT1expression and clinical pathological characteristics;5, Lentivirus expressing SIRT1-target shRNA was produced by plasmid. Gastric cancer cell lines and gastric epithelial cells were infected by lentivirus. Fluorescence microscope and immunoblotting were used to confirm the validation of lentivirus and infection.6, CCK-8analysis was used to test the effect of shRNA-SIRT1on proliferation of gastric cancer cells and gastric epithelial cells;7, APC-Annexin V analysis and flow cytometry were used to test the effect of shRNA-SIRT1on apoptosis of gastric cancer cells and gastric epithelial cells;8, STAT3expression in shRNA-SIRT1infected gastric cancer cells and control group were detected by real time PCR and immunoblotting.9, Co-immunoprecipitation was used for analyzing the relationship between SIRT1and STAT3, PSTAT3in gastric cancer;10, STAT3knock out mice model was successfully built.11, SIRT1expression in gastric cancer tissue were detected by the method of immunohistochemical staining in STAT3-KO mice.Results:1, SIRT1mRNA expression in the21fresh gastric cancer tissue is higher than non-tumor mucosa.The mRNA and protein levels of SIRT1in all three gastric cancer cells are higher than in epithelial cells.Through the immunohistochemical staining on130gastric cancer cases, we found the SIRT1expression was higher in carcinoma than normal mucosa.The SIRT1expression was positively corelated with the depth of tumor invasion, lymphatic metastasis, TNM stage and tumor size and was negatively corelated with the survival time. Although the SIRT1expression was higher in10young cases, SIRT1was also positively corelated with age.2, Lentivirus expressing SIRT1-target shRNA was produced successfully by plasmid. Gastric cancer cell lines and gastric epithelial cells were successfully infected by lentivirus. Comparing shRNA-SIRT1group with control, we found that shRNA can suppress the gastric cancer cells proliferation and promote the apoptosis by silencing SIRT1, nearly no effect on gastric epithelial cells.3, There was no obvious difference of STAT3mRNA level between shRNA-SIRT1group and control, but we found the protein level of STAT3was higher on gastric cancer cell group and control by immunoblotting analysis.P-STAT3was same with STAT3, but the A-STAT3was opposite. The SIRT1could form complexes with STAT3and P-STAT3by co-immunoprecipitation.4, After analyzing the SIRT1expression in STAT3knock out mice gastric cancer model,we found the SIRT1was lower in STAT3-KO group.It indicated STAT3interacted with SIRT1in gastric cancer.Conclusion:As a NAD+-dependent deacetylase, SIRT1plays an important regulating role in cell survival, senescence and apoptosis. But there was a controversy about whether SIRT1was oncogene or anti-oncogene.Through affirming the high expression in gastric cancer tissue and cell,we consider SIRT1as a oncogene in gastric cancer.Applying SIRT1-target shRNA technique for suppressing the proliferation and promoting the apoptosis of gastric cancer cell, we conclude that SIRT1play a part in gastric cell proliferation and apoptosis, providing a experimental basis for novel biological gene therapy.We consider that SIRT1may indirectly increase the phosphorylation STAT3by deacetylating STAT3and form a complex, promoting the progress of gastric cancer.Through acting at JAK1-STAT3or activating STAT3directly, SIRT1participates in the development of gastric cancer.The project provides a theory evidence and experimental basis for further studying of the molecular mechanism and signal channel for SIRT1. |
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