The Mechanism Of Overexpression Of SPARC Enhances Chemosensitivity In Peritoneal Metastasis Of Gastric Cancer

Introduction: Gastric cancer is one of the most common cancers in the digestive system worldwidely.Because its early symptoms are not obvious,about 25% of gastric cancer patients are diagnosed with peritoneal metastasis before operation or during operation,thereby losing the chance of radical operation.Effective prevention,early diagnosis and timely treatment of peritoneal metastasis have been the research difficulties and hot spots in the field of gastric cancer treatment.Therefore,it is important to study the molecular mechanism of the development of gastric cancer and peritoneum metastasis,and explore new therapeutic ways and targets to improve the prognosis of gastric cancer.The SPARC gene is a single copy gene,highly conserved,with more than 70% of the amino acid sequence homologous in various organisms.It is located on the human chromosome 5q31.3-q32,which encodes acidic matrix related protein rich in cysteine.The protein can resist cell adhesion,regulate cell proliferation and regulate tissue differentiation,and it has a great relationship with tumor proliferation,invasion and metastasis.Objective: The purpose of this study was to investigate the expression of SPARC in gastric cancer cell lines,the molecular regulation mechanism,and explore its biological function,the combined application of DNA methyltransferase inhibitor(5-Aza-2-deoxyctidine)and chemotherapeutic drugs(5-FU,CDDP)cell lines and gc9811-p drug intervention on gastric cancer peritoneal metastasis animal model,feasibility to investigate the SPARC as the peritoneal metastasis of gastric cancer peritoneal chemotherapy target.Method: Real time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression difference of SPARC in 4 gastric cancer cell lines and normal human gastric epithelial cell line GES-1,and the results were statistically analyzed.Methylation specific PCR(MSP)was used to detect the methylation status of gastric cancer cells(MKN-45)and normal human gastric mucosal epithelial cell line(GES-1).In vitro experiment: in MKN-45 cell line,lentivirus was transfected,overexpressing SPARC was overexpressed,and the effect of over expression of SPARC on biological function of gastric cancer cells was analyzed by CCK-8,scratch healing test,flow cytometry and Transwell chamber assay.In vivo experiment: to construct a stable cell line expressing SPARC and establish a model of ascites in nude mice,and to analyze the effect of overexpression of SPARC on the growth of abdominal metastases in nude mice.In vitro experiment: in the MKN-45 cell line,the exogenous overexpression of SPARC was combined with chemotherapeutic drugs,combined with demethylation drugs and chemotherapeutic agents.The effects of different drug intervention on the biological function of gastric cancer cells were analyzed by CCK-8 and flow cytometry.In vivo experiment: the model of the ascites in nude mice was established,and the effects of the drug on the growth of the tumor in the abdominal cavity of nude mice were analyzed by different drug intervention.Result: 1.Compared with normal human gastric epithelial cell line GES-1,the expression level of SPARC in MKN-45 cell line was significantly lower than that in normal gastric epithelial cell line.2.Methylation specific PCR(MSP)results showed that in normal human gastric epithelial cell line GES-1,the promoter of SPARC gene showed partial methylation status,while in MKN-45 cell lines,the promoter of SPARC gene showed hypermethylation status.The gastric cancer cell line was intervened with the demethylation drug 5-Aza-d C.The results of q RT-PCR showed that the expression level of SPARC was significantly higher.3.In vitro transfection of lentivirus to MKN-45 cell lines,CCK-8 results showed that overexpression of SPARC could inhibit the proliferation of MKN-45 cells.Flow cytometry showed that overexpression of SPARC could significantly promote the apoptosis of MKN-45 cells.The scratch test and Transwell laboratory test showed that overexpression of SPARC could inhibit the migration and invasion of MKN-45 cells.A SPARC overexpressed nude mouse model was constructed,and the results showed that overexpression of SPARC could inhibit the growth of metastatic carcinoma nodules in nude mice.4. MKN-45 cell lines were treated with different chemotherapeutic drugs.The results of CCK-8 showed that single application of chemotherapeutic agents(5-FU and CDDP)could inhibit the proliferation of MKN-45 cell lines.Overexpression of SPARC combined with chemotherapeutic drugs could further inhibit the proliferation of MKN-45 cell lines.The nude mouse metastasis model of ascites,demethylation drugs combined with chemotherapy drug intervention,results show that to chemotherapy drugs can be methylated indirectly by overexpression of SPARC can inhibit the proliferation of MKN-45 cells,further inhibition of ascites formation and intraabdominal cancer nodules metastasis,prolong the survival time of mice.Conclusion: 1.The expression of SPARC gene is low in gastric cancer cell line and plays a role in the tumor suppressor gene.The downregulation expression of SPARC gene in gastric cancer cell line is related to the hypermethylation status of SPARC gene promoter region,and the expression of SPARC can be restored by using the demethylation drug(5-Aza-d C).2.Overexpression of SPARC gene can inhibit the proliferation,invasion and metastasis of gastric cancer cell,and promote its apoptosis.3.Overexpression of SPARC can effectively improve the sensitivity of chemotherapy drugs,combined application of demethylation drugs 5-Aza-d C and 5-FU chemotherapy or CDDP can indirectly increase the sensitivity to chemotherapy and provide new therapeutic targets and treatment methods for peritoneal metastasis of gastric cancer intraperitoneal chemotherapy.