| [Background]Gastric cancer（GC）is a highly invasive and lethal digestive system malignant tumor.The invasion and metastasis of gastric cancer is the main reason for failure treatment.Previous studies found that the epithelial mesenchymal transition（EMT）is closely related to the malignant biological behavior of tumor.Recent studies have found that silent information regulator-1（SIRT1）plays an important role in the pathogenesis of gastric cancer.Our previous studies have shown a certain correlation between the expression level of SIRT1 and the formation of EMT in gastric cancer.In order to explore the role of SIRT1 in mediating EMT and promoting malignant phenotype of gastric cancer,our study aims to elucidate the correlation between SIRT1 and EMT,prognosis and malignant biological behavior of gastric cancer patients,and its possible molecular mechanism,so as to explore effective new targets for diagnosis and treatment of gastric cancer and to provide experimental theoretical basis.Chapter 1: The correlation among SIRT1 expressionand EMT and the clinicopathological characteristics of gastric cancer[Objective]:In order to determine the expression of SIRT1 and EMT level in gastric cancer tissues,and evaluate the correlation among SIRT1 expressionand EMT leveland clinical prognosis.[Methods]:1.105 cases gastric cancer tissues and corresponding adjacent tissues were collected.The baseline clinical data were collected.HE staining was used to determine the pathological classification and histological differentiation of gastric cancer.2.The expressions of SIRT1,H3K9me3,H3K9 Ac,vimentin and E-cadherin in gastric cancer tissues and corresponding adjacent tissues were detected by immunohistochemistry.3.Clinical follow-up was completed and the relationshiop among the expressions of SIRT1,H3K9me3,H3K9 Ac,vimentin and E-cadherin and the clinicopathological characteristics in68 cases of gastric cancer were analyzed.4.Analyze the correlation of SIRT1,H3K9me3,vimentin and E-cadherin in 68 cases of gastric cancer and their correlation of clinical prognosis.[Results]:1.In the clinical data of patients with gastric cancer,there are more male patients than female patients and moderately differentiated adenocarcinoma is the main histological type.The majority of gastric cancer was advanced with the serosal layer or extraserosal layer involved at initial diagnosis.Most patients had lymph node metastasis.Compared with the adjacent tissues,SIRT1,H3K9me3 and vimentin were highly expressed in gastric cancer tissues;Ecadherin was expressed both in gastric cancer tissues and adjacent tissues with the strong positive expression rate of 45.71% and the weak positive expression rate of 54.29% in gastric cancer tissues,and the strong positive expression of 100% in adjacent tissues;and H3K9 Ac was lower expressedboth in gastric cancer tissues and adjacent tissues.2.The expression of SIRT1,H3K9me3 and vimentin was positively correlated totumor invasion depth,lymph node metastasis,TNM stage and survival time（P<0.05）;but the weak positive expression of E-cadherin was correlated to tumor invasion depth,tumor size,lymph node metastasis,TNM stage and survival time（P<0.05）;There was no correlation between the expression of H3K9 Acand tumor size,differentiation,depth of invasion,lymph node metastasis and TNM stage（P>0.05）.3.Spearman correlation analysis showed that SIRT1 was positively correlatedto H3K9me3andvimentinexpression（P<0.05）;but negatively correlated to E-cadherin expression（P<0.001）.Meanwhile,highly expression of SIRT1 and H3K9me3 and the low expression of E-cadherin were positively correlated to oor prognosis（P<0.05）.The expression of H3K9 Ac was not correlated to the prognosis of gastric cancer（P>0.05）.[Conclusion]:There is a correlation between SIRT1 and EMT in gastric cancer tissues.The highly expression of SIRT1 is positively correlated to the tumor invasion depth,lymph node metastasis,TNM stage and poor prognosisof gastric cancer,suggesting that SIRT1 may participate in EMT and lead to poor prognosis of gastric cancer.Chapter 2: SIRT1 mediates EMT formation and regulates malignant biological behavior of gastric cancer cells[Objective]:In oreder to evaluate whether SIRT1 is involved in the formation of EMT in gastric cancerand further explore the influence of SIRT1 on EMT of gastric cancer cells.[Methods]:1.Use one normal gastric mucosal cell lines as a control to screen poorly differentiated cell lines from three different differentiated gastric cancer cell lines.The effects of different time and different doses of TGF-b1 on cell proliferation and SIRT1 expression are detected by CCK-8 and Western Blot.2.SIRT1 expression in SIRT1 si RNA knockdown gastric cancer cell lines was established.Whetherknockdown of SIRT1 could reverse the formation of EMT and the changes in cell proliferation,invasion and metastasis of gastric cancer cells induced by TGF-b1 was detected by CCK-8,Transwell,cell scratch and Western Blot.3.The subcutaneous tumorigenesis model of nude mice was established using gastric cancer MNK-45 cell line.The growth curve and weight change of tumor were recorded without intervention.The regulating effect of TGF-b1 induction or SIRT1 konckdown on EMT in gastric cancer tumor-bearing mice was detected by immunohistochemistry,and the correlation between SIRT1 and EMT was analyzed.[Results]:1.Both MNK-45 and BGC-823 cells have high expression of SIRT1 and vimentin（P<0.05）and low expression of E-cadherin（P<0.05）compared to normal gastric mucosal cell lines;TGF-b（16）（5ng/ml）induced the expression of SIRT1 in MNK-45 and BGC-823 cells is in a time-dependent manner（24-48h）and promotes cell proliferation（P<0.05）.2.After SIRT1 expression was induced by TGF-b（16）（11）the results of CCK-8,Transwell and cell scratch test showed that SIRT1 knockdown inhibited proliferation,invasion and metastasis of MNK-45 and BGC-823 cells significantly（P<0.05）;Western blot results showed that SIRT1 knockdown inhibited the expression of vimentin and upregulate the expression of E-cadherin（P<0.05）.3.Vivo experiments showed the significant increased tumor volume of the tumor-bearing mice induced by TGF-b（16）and the increased weight of tumor tissue compared to the control group（P<0.01）;However,the tumor volume of SIRT1 konckdown tumor-bearing mice was significantly decreased and the weight of tumor tissue was significantly reduced compared to the control group（P<0.01）.4.Immunohistochemical results showed that TGF-b（16）increased the expression of SIRT1,H3K9me3,vimentin and decreased the expression of E-cadherin in tumor bearin mice.Knockdown of SIRT1 inhibited the expression of SIRT1,H3K9me3 and vimentin,and upregulated the expressionof E-cadherin.[Conclusion]:Both in vitro and in vivo results showed that the expression of SIRT1 induced by TGF-b1in MNK-45 and BGC-823 cells is in a time-dependent manner（24-48h）and promotes cell proliferation;SIRT1 konckdown inhibited the expression of vimentin and upregulated the expressionof E-cadherin,inhibited proliferation,invasion and metastasis of cancer cells,indicating SIRT1 mediates the formation of EMT in gastric cancer cells and regulates the malignant biological behavior of gastric cancer cells.Chapter3: he mechanism of circ RNANOP10/mi R-204/SIRT1 axis regulating EMT in gastric cancer[Objective]:In order to investigate the effects of circ RNANOP10/mi R-204/SIRT1 axis on EMT of gastric cancer and its molecular mechanism.[Methods]:1.Based on high-throughput chip screening technology,we aimed to explore the characteristics of circ RNA expression in gastric cancer and adjacent tissues.Real-time PCR was used to detect the expression of circ NOP10,and SIRT1 m RNA in plasma of patients with gastric cancer.2.RNAhybrid and Target Scan were used to predict the regulatory relationship among circ NOP10,mi R-204 and SIRT1.The relationship between circ NOP10 and mi R-204 was verified by pull-down and in situ hybridization.3.The expression of SIRT1,vimentin and E-cadherin in MNK-45 and BGC-823 cells was detected by Western Blot.Circ NOP10 cell models were establishedand CCK-8 and FCM were used to determine the effects of SIRT1 gene on the biological behavior of gastric cancer.4.The relationship among circ NOP10、mi R-204 and SIRT1 was verified by gene knockout and lentiviral transfection technology.[Results]:1.The hsacirc0034351 screened by circRNA was located on chromosome 15.NOP10 gene in exon 2 was the most up-regulated gene in gastric cancer,which was designated as circ NOP10.Moreover,the expression of circ NOP10 and SIRT1 m RNA in plasmaof patients with gastric cancer was significantly up-regulated compared to the control group（P<0.01）.2.Pull down and in situ hybridization experiments confirmed that there was a binding site between circ NOP10 and mi R-204.The expression of circ NOP10 in poorly differentiated gastric cancer MNK-45 and BGC-823 cells was higher than that in moderately differentiated HCG-27 cells（P<0.05）.3.In MNK-45 cells,the knockdown of circ NOP10 inhibited the expression of circ NOP10 and SIRT1 m RNA,decreased the expression of vimentin and increased the expression of Ecadherin（P<0.05）;It also affected the invasion and metastasis ability of gastric cancer（P<0.05）.The similar phenomenon was verified in poorly differentiated gastric cancer cell line BGC-823（P<0.05）.4.Transfection of anti-mi R-204 lentivirus into gastric cancer cells up-regulated the expression of SIRT1 and Vimentin,and inhibited the expression of E-cadherin（P<0.05）;Meanwhile,knockdown of circ NOP10 and anti-mi R-204 down-regulated the expression of SIRT1 and Vimentin,and up-regulated the level of E-cadherin（P<0.05）.[Conclusion]:Circ NOP10 / mir-204 / SIRT1 axis mediates the EMT formationin gastric cancer cells,and then affects the malignant biological behavior of gastric cancer cells.SIRT1 may be a key molecule in the EMT formation in gastric cancer. |
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