| BackgroundGastric cancer(GC)is one of the most common tumors in the world.China is a region with high morbidity and mortality.Early detection,timely treatment can significantly improve the survival of many tumor patients,including gastric cancer.But because of the hidden symptoms,the difficulty of screening,as well as the rapid progress of gastric cancer,strong invasive ability and so on,the incidence of gastric cancer metastasis remains high,and the prognosis is poor.Most gastric cancer was diagnosed as advanced,peritoneal metastasis is a common type of metastasis.Peritoneal metastasis of gastric cancer has always been the bottleneck of the therapeutic effect of gastric cancer,and we still lack of research in this area.Therefore,there is an urgent need to study the potential mechanism and characteristics of peritoneal metastasis of gastric cancer,so as to provide new targets and interventions for the diagnosis,treatment and prognosis of gastric cancer.ObjectiveOvercoming oxidative stress and resisting anoikis is very important for tumor growth and metastasis.In order to understand the resistance anoikis of gastric cancer and antioxidation metabolism,and to find the related therapeutic target.The main purpose of this project is to: 1.To explore the effect of Nicotinamide nucleotide dehydrogenase(NNT)on NADPH homeostasis regulation and anoikis resistance in gastric cancer.2.In the microenvironment of adipocytes/gastric cancer cells,the metabolic remodeling of cancer cells,accumulation of lipid droplets through diacylglycerol acyltransferase 2(DGAT2)to resistance anoikis and realize the function of peritoneal metastasis.MethodsPart 11.RT-q PCR and WB were used to detect the expression of NNT m RNA and protein in GC cells and tissue specimens.The expression of NNT in gastric cancer specimen was detected by IHC,and the survival curve was drawn according to clinical data.2.Construction of NNT knockdown gastric cancer cell lines(HGC27,BGC823)by lentivirus.After suspension culture,NADPH/NADP+ and GSH/GSSG of gastric cancer cells in NNT knockdown group and control group were detected.Flow cytometry was used to detect mitochondrial and total ROS levels and anoikis after Annexin V/PI staining.Soft agar colony formation assay was used to detect the anchorage-independent growth ability of gastric cancer cells.3.The NNT knockdown gastric cancer stable cell line and the control cell line were injected into the nude mice by subcutaneous inoculation,intraperitoneal injection and caudal vein injection to observe the growth of subcutaneous tumor and the metastasis of mesenteric tumor nodule and pulmonary metastasis nodule.The specimens were embedded and stained to observe the pathology.Part 21.High fat diet(HFD)induced obesity model in mice(nude mice,SCID mice),and monitored body weight and blood lipid.To observe the effect of obesity or HFD on peritoneal metastasis of gastric cancer.The m RNA expression of key enzymes of fatty acid metabolism pathway in abdominal tumor nodule of mice in common diet group and HFD group was compared.2.Human peritoneal omental adipocytes were extracted and co cultured with gastric cancer cells.The m RNA and protein expression of DGAT2 were detected.The content and distribution of lipid droplets in the cells were observed by BODIPY 493/503 fluorescent staining.3.Construction of DGAT2 knockdown gastric cancer cell line by lentivirus.After coculture with adipocytes,the lipid drop content in the control group and DGAT2 knockdown group was observed by fluorescence staining.The contents of triglyceride,NADPH/NADP+ and GSH/GSSG were measured.4.Under the condition of suspension culture,the cell level and ROS content of mitochondria of gastric cancer cells cocultured with adipocytes was measured.The anoikis of gastric cancer cells was detected by Calcein AM/Eth D-1 staining.5.DGAT2 knockdown gastric cancer stable cell line and control cell line was injected into nude mice and SCID mice.To observe the peritoneal metastasis and lung metastasis in mice with HFD.6.After pretreatment of gastric cancer cells with DGAT2 inhibitor,the lipid drop content of cells in control group and inhibitor group was observed.Under the condition of suspension culture,the anoikis of gastric cancer cells and adipocytes after co culture was detected;at the same time,the intraperitoneal tumorigenesis of mice was observed after intraperitoneal injection of inhibitor.7.The protein expression of DGAT2 was detected by IHC in human gastric cancer,para-carcinoma tissues and lymph node metastasis tissues.WB was used to detect the protein expression level of DGAT2 in matched gastric cancer tissue specimens.Combined with clinical data,the survival curve of prognosis was drawn and Cox regression model was used to analyze the prognostic factors.ResultsPart 11.The expression of NNT m RNA and protein was increased in gastric cancer cells or tissues.NNT was highly expressed in human gastric cancer and lymph node metastasis.Survival analysis showed that the high expression of NNT was negatively correlated with OS(P =0.003)and DFS(P =0.006).2.Successful construction of NNT knockdown gastric cancer cell lines(HGC27,BGC823).After suspension culture,the contents of NADPH/NADP+ and GSH/GSSG decreased significantly in NNT knockdown group(P <0.01).The level of ROS in mitochondria and cells increased,the anoikis of GC cells increased,and the ability of anchorage-independent growth decreased.3.The results of animal experiments showed that the ability of subcutaneous tumorigenesis of gastric cancer cells in NNT knockdown group decreased,the cell proliferation was decreased and apoptosis was increased.The lung metastasis and peritoneal dissemination were inhibited in knockdown group,and the survival time of the mice was longer(P <0.01).Part 21.HFD induced obesity model and intraperitoneal tumorigenesis model were established successfully.The mice in HFD group had more intraperitoneal disseminated tumors than those in normal diet group,and the survival time of mice(nude mice)was significantly shorter(P =0.017).The expression of CD36,GPD1/2,GPAT1,AGPAT1, PAP,DGAT1/2,CPT1 A,NADK2 and NNT were significantly higher in the abdominal metastatic tumor nodes than in the control group(P <0.01).The expression of DGAT2 increased most obviously.2.Adipocytes were successfully extracted.After coculture with gastric cancer cells,the expression of DGAT2 m RNA and protein increased,and the content of lipid droplets increased.3.DGAT2 knockdown gastric cancer cell line was successfully constructed.After coculture,the amount of lipid droplet formation in knockdown group was less than that in the control group,and the content of TG,NADPH/NADP+ and GSH/GSSG in cells decreased(P <0.01).4.In suspension culture,after coculture with adipocytes,the level of mitochondria and total ROS of gastric cancer cells decreased,the anoikis of GC cells decreased.However,these phenomena can be reversed by DGAT2 knockdown.5.The results of animal experiments showed that DGAT2 knockdown significantly inhibited the peritoneal tumorigenicity of HFD induced nude mice and SCID mice(P <0.01)and lung metastasis in nude mice(P <0.05).6.After pretreatment with DGAT2 inhibitor,the formation of lipid droplets in gastric cancer cells induced by oleic acid was inhibited.In suspension culture,the anoikis of gastric cancer cells in inhibitor group increased.After intraperitoneal injection of inhibitor,the tumorigenicity of mice was weaker than that of the control group.7.IHC results showed that DGAT2 was highly expressed in lymph node metastasis and omental metastasis of gastric cancer.The level of DGAT2 protein in the matched metastasis tissue was higher than that in the primary tumor tissue.Survival analysis showed that OS and DFS of patients with high DGAT2 expression were shorter than those with low expression(P <0.001).In addition,Cox regression analysis of survival data showed that DGAT2 was highly expressed(RR =1.50,95% CI: 1.01-2.21,P =0.043),lymph node invasion(RR =2.08,P =0.021)and advanced gastric cancer(RR =2.47,P =0.000)were independent adverse prognostic factors.ConclusionPart 11.The overexpression of NNT is positively correlated with metastasis and poor prognosis of gastric cancer.2.Knockdown of NNT resulted in a significant decrease in NADPH and an increase in ROS levels of GC cells,and accelerated the anoikis of gastric cancer cells under the stress of anchorage-independent growth.3.Knockdown of NNT inhibited the growth,lung metastasis and peritoneal dissemination of gastric cancer cell transplanted tumors in nude mice.Part 21.HFD promotes peritoneal metastasis of gastric cancer and upregulates DGAT2 expression in mice.2.DGAT2 promotes lipid droplet formation and NADPH production induced by adipocytes or oleic acid.3.DGAT2 regulates redox homeostasis and enhances the resistance anoikis of GC cells.4.Knockdown of DGAT2 inhibited the HFD-induced peritoneal dissemination and lung metastasis of gastric cancer in mice.5.Gastric cancer patients with high DGAT2 expression have poor prognosis. |
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