| 【Background】Gastric cancer is the fifth most common cancer in the world and the third most common cause of death in the world.Gastric cancer is also one of the most common diseases in China.The 5-year survival rate of patients with gastric cancer is 30%.Although many biomarkers related to gastric cancer have been studied,gastric cancer are usually diagnosis as the advanced stage in clinical.However,gastric cancer patients with the same stage of primary tumor,regional lymph node and distant metastasis(TNM)often show different clinical symptoms.In this paper,we study the metabolites and lipids of gastric cancer,the key factors affecting the development of gastric cancer and its potential therapeutic targets by using lipomics analysis.Recently,fatty acid(FA)metabolism has been reported to be of great significance in the occurrence and development of a variety of cancers both in vivo and in vitro.Many studies have suggested that the abnormal expression of genes related to fatty acid synthesis or oxidation was related to tumor metastasis,drug resistance and recurrence.The imbalance between fatty acid synthesis and oxidation might lead to insufficient fatty acid levels and lipid accumulation.From the results of lipomics analysis,we found that the expression level of Palmitic Acid,which is a saturated fatty acid in fatty acid,decreased in gastric cancer.Further study the potential role of Palmitic Acid in clinical gastric cancer tissues and the potential related molecular mechanisms.Signal transducer and activator of transcription-3(STAT3)was initially considered as a potential cytoplasmic transcription factor that could be activated to regulate inflammation and immunity.Nowadays,STAT3 has been considered to be a pluripotent transcription factor in various cancers,which could act as the oncogenes by activating genes involved in cell differentiation,proliferation,apoptosis,metastasis,angiogenesis and metabolism.Different from other STATs family proteins,STAT3 contains six distinct domains,including NH2-terminal domain,crimp coil domain,DNA binding domain,junction domain,Src homology 2(SH2)domain and transcriptional activation domain.Its transcriptional activity depended on the phosphorylation of Tyr705 in the transcriptional activation region.Phosphorylated STAT3 could interact with other STAT family proteins to form homodimers or heterodimers through the interaction of tyrosine phosphorylation of SH2,and translocated into the nucleus to activate gene transcriptional activity.PIAS3 was clearly reported as the endogenous inhibitor of STAT3 and was involved in the occurrence and development of several cancers.However,the potential mechanism of Palmitic Acid on affecting the growth and metastasis of gastric cancer cells by regulating the expression levels of p-STAT3 and PIAS3 is still unclear.The purpose of this study was to explore the role and the potential mechanism of Palmitic Acid in human gastric cancer cells.We have been applied UPLC-MS/MS technique to lipogenic analysis and found that Palmitic Acid was down-regulated in human gastric cancer tissues.Furthermore,150μM Palmitic Acid could inhibit the cell proliferation and invasion,and induced cell apoptosis of gastric cancer cells.In addition,we found that Palmitic Acid could inhibit the expression levels and the nuclear translocation of p-STAT3,and inversely regulated the expression levels of PIAS3 in human gastric cancer cells.The study of the potential mechanism of Palmitic Acid on inhibiting the development of gastric cancer is expected to provide new ideas for the study of lipid metabolism and prevention and treatment of gastric cancer.【Research methods】(1)The effects of Palmitic Acid on the proliferation of human gastric cancer cells was detected by MTT assay,plate cloning assay and Ed U-DNA-assay.The effects of Palmitic Acid on the invasion of gastric cancer cells were detected by Transwell invasion test.(2)Annexin-V-FITC/PI dual-staining assays were used to detect the apoptosis of human gastric cancer cells after Palmitic Acid treated.(3)To estimate the relationship between survival time and prognosis of patients with STAT3,PIAS3 expression in gastric cancer was studied by using Kaplan Meier analysis.(4)Western blotting(WB)and Real-Time quantitative PCR(qRT-PCR)were used to detect the expression levels of p-STAT3,PIAS3,p-JAK2,N-cadherin and Vimentin in human tissues or cell lines after Palmitic Acid treatment.(5)H&E staining was used to detect cancer tissues from paracancerous tissues,and immunohistochemical(IHC)staining was used to detect the expression levels of p-STAT3 in human gastric cancer tissues.(6)Immunofluorescence(IF)staining was used to detect the expression level and nuclear translocation of p-STAT3 after Palmitic Acid treated.【Research results】(1)The results of MTT assay(24h,48 h and 72h)showed that the proliferation inhibitory effects of Palmitic Acid on the four human gastric cancer cell lines were more sensitive than that of human normal gastric epithelial cells and human normal hepatocytes with concentration-dependent manners.The results of plate cloning(BSA,50μM,100μM,200μM)showed that 200μM Palmitic Acid significantly inhibited the colony formation of gastric cancer cells with the inhibitor.Ed U-DNA incorporation assay also showed that 50μM Palmitic Acid could inhibit human gastric cell proliferation with the corresponding inhibition rate range from 80.0% to 30.6%.The results were consistent with those of MTT and plate cloning test.Cell Transwell invasion test showed that the inhibitory effect of Palmitic Acid on cell invasion was concentration-dependented manner.(2)FCM analysis showed Palmitic Acid induced human gastric cancer cell apoptosis after Palmitic Acid treated for18 h.(3)Survival curve analysis showed that the expression levels of STAT3,PIAS3 were closely related to the prognosis of patients with gastric cancer in clinical.(4)The expression levels of p-STAT3,p-STAT3 and p-JAK2 were higher in human gastric cancer cells and human gastric cancer tissues in clinical,while the expression levels of PIAS3 was lower.Furthermore,When Palmitic Acid was added to gastric cancer cells,the expression levels of p-STAT3 and p-JAK2 were decreased compared with the control group after treated with Palmitic Acid for12 h,While the expression levels of PIAS3 was higher than that in the control group.In addition,the expression levels of N-cadherin and Vimentin were lower than that in the control group after Palmitic Acid treated for12 h.(5)H&E staining showed that there were a better distinction between gastric cancer tissues and paracancerous tissues.Immunohistochemistry staining showed that the expression levels of p-STAT3 was higher in human gastric cancer tissues.(6)Palmitic Acid inhibited the expression levels of p-STAT3 in the nuclear of human gastric cancer cells.【Conclusions】(1)Palmitic Acid inhibits the human gastric cells proliferation and invasion.(2)Palmitic Acid induced cell apoptosis in human gastric cancer cells.(3)STAT3 and PIAS3 were closely related to the prognosis of patients with gastric cancer in clinical.(4)The expression levels of p-STAT3 and p-JAK2 were higher in human gastric cancer tissues in clinical and in human gastric cancer cell lines than in human gastric paracancerous tissues in clinical and in human gastric normal cell lines,while the expression levels of PIAS3 were lower.(5)Palmitic Acid inhibited the expression levels of p-STAT3,p-JAK2,N-cadherin and Vimentin,while inversely regulated the expression levels of PIAS3 in human gastric cancer cells.(6)Palmitic Acid inhibits the nuclear expression of p-STAT3 in gastric cancer cells. |
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