| BackgroundsGastric cancer(GC)is one of the most common malignant tumor and the second common cause of cancer-associated death worldwide.No effective treatment modalities are available for patients with advanced gastric cancer in whom the five-year survival rate was reported to be around 20-30% at present.Although many studies identifying the molecular markers in gastric cancers via proteomic technology have been reported previously,the proteomic study screening the molecular markers during the typical process of the gastric intraepithelial neoplasia including normal gastric antrum mucosa,atrophic gastritis of mild dysplasia,moderate dysplasia,severe dysplasia and early gastric cancer)has not been conducted to date.AimsWe examined the differentially expressed proteins among the normal gastric antrum mucosa,atrophic gastritis of mild dysplasia,moderate dysplasia,severe dysplasia and early gastric cancer,and evaluated their potential biological functions to further explore the carcinogenesis mechanism of gastric cancer,and then develop novel early diagnostic methods and therapeutic targets.Methods1)A total of 160 specimens(10% neutral buffered formalin fixed paraffin embeded)of different-staged gastric tissues were enrolled in this study,including 30 cases of normal gastric antrum mucosa,30 cases of atrophic gastritis of mild dysplasia,30 cases of moderate dysplasia,30 cases of severe dysplasia and 40 cases of early gastric cancer.2)The targeted tissues were extracted using microdissection and the expression profiles of total proteins were detected by lable-free quantification technology integrated with liquid chromatography-tandem mass spectrometry(LC-MS/MS).Proteins with significant differential expression were analyzed by bioinformatics analysis using the Maxquant software with Gene ontology enrichment and KEGG pathway.The results were verified by immunohistochemistry in tissue array.3)Lentivirus-mediated gene transfer was used to overexpress ERGIC1 in SGC-7901,BGC-823 gastric cancer cells.Cell viability was measured by MTT assay and apoptosis were determined by flow cytometry.4)Association between FAM83 D expression and clinicopathologic factors was evaluated by immunohistochemistry in 291 gastric cancer specimens.5)Overexpress and depletion of FAM83 D in vitro were constructed in SGC-7901 and AGS cell models.Meanwhile,cell viability of both cells was measured by MTT assay.Cell cycle progression and apoptosis were determined by flow cytometry.Cell colony-forming capacity was measured by colony formation assay,and migration and invasion capacity were determined by transwell assay.6)Using xenograft model in nude mice to test whether knockdown of FAM83 D influenced the tumorigenicity of cancer cells in vivo.7)Western blot analysis was applied to determine whether knockdown and up-regulation of FAM83 D affected the activity of Wnt/?-catenin pathway.Results1)The results of LC-MS/MS showed that 355 proteins expressed differently among the five groups(>1.2-fold change and a p value <0.05 are considered significant).2)Among those 355 proteins expressed,we found that the expression of ERGIC1 decreased gradually but DNA-PKcs expression increased in gastric mucosa as its pathological morphology change from normal to severe epithelial dysplasia and then gastric cancer.Furthermore,the expression differences of ERGIC1 and DNA-PKcs were verified by the immunohistochemical results of tissue chip with 150 samples from our center.3)With the overexpression of ERGIC1 in human GC SGC-7901 and BGC-823 cells,the process of proliferation and apoptosis of both cells was inhibited and promoted,respectively.4)From the results of LC-MS/MS,we found 55 proteins had significantly differential expression between the normal gastric mucosa and early gastric cancer(>2-fold change and a p value <0.05 are considered significant).In particular,the results of LC-MS/MS showed that one of the most significantly overexpressed proteins was FAM83 D in gastric cancer compared with the normal gastric mucosa,which was further confirmed by the immunohistochemical results of tissue chip with 291 samples from our center.Furthermore,the Kaplan-Meier analysis on the 291 cases from our center,coupled with the information obtained from the Oncomine database,indicated that the FAM83 D expression was inversely correlated with overall survival and disease-free survival in patients with gastric cancer,and we also observed that FAM83 D overexpression was related to larger tumor size,poor differentiation and more frequent distant metastasis.5)Down-regulation of FAM83 D in human gastric cancer AGS and SGC-7901 cells decreased the process of proliferation,migration and invasion,and increased the percentage of G1-phase cells but decreased the percentage of S-phase cells.On the contrary,overexpression of FAM83 D in human gastric cancer cells enhanced the ability of proliferation,apoptosis,migration and invasion,lessened the percentage of G1-phase cells but increased the percentage of S-phase cells.6)Silencing of FAM83 D gene inhibited the tumorigenicity of gastric cancer cell in nude mice model.7)Western blot analysis showed that p-GSK-3? expression levels were significantly increased,but the expressions of ?-catenin,cyclinD1,and c-Myc were substantially down-regulated when FAM83 D gene was silenced in AGS and SGC-7901 cells;Inversely,p-GSK-3? expression level was significantly decreased,but the expressions of ?-catenin,cyclinD1,and c-Myc were enhanced obviously when FAM83 D was overexpressed in AGS and SGC-7901 cells.ConclusionsThere were many proteins expressed differently among the normal gastric antrum mucosa,atrophic gastritis of mild dysplasia,moderate dysplasia,severe dysplasia and early gastric cancer,which might be expected to be novel early diagnostic and targeted therapeutic biomarker.DNA-PKcs and ERGIC1 expression might be involved in the initiation of gastric cancer.They might be potential oncogene and a suppressor gene in gastric cancer,which need further to be validated in future.FAM83D expression was inversely correlated with overall survival and disease-free survival in patients with gastric cancer,and we also observed that FAM83 D overexpression was related to larger tumor size,poor differentiation and more frequent distant metastasis.FAM83 D might be involved in cell proliferation and invasion in gastric cancer via activation of the Wnt/?-catenin signaling pathway.In summary,FAM83 D might be expected to be a promising diagnostic and therapeutic target for human gastric cancer. |
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