Clinical Significances And Biological Function Of Up-regulated Trans1640brane Protein GRINA In Gastric Cancer

Objective: By conducting bioinformatics analysis of the commonly used gastric cancer databases and clinical verification of gastric cancer tissue samples,combined with biological function both in vitro and in vivo,to determine the relationship between the expression of up-regulated gene GRINA and gastric cancer progression.To explore the molecular mechanism of GRINA up-regulation and its function in the carcinogenesis and progression of gastric cancer.Methods:Through analysing several international authoritative databases like the GEO database,ONCOMINE database,TCGA database which contain large sample of gastric cancer and normal tissues,by comparing the different expression of the genes in cancer and normal tissues,gene GRINA expression was found significantly up-regulated in gastric cancer tissues compared with normal tissues.2.The mRNA and protein in 20 paired fresh gastric cancer and normal tissues were extracted,the expression of GRINA in gastric cancer tissues and normal tissues was compared by RT-PCR and Western blot(Western blotting).3.Paraffin beded specimens of gastric cancer were collected for microarrary construction.Immunohistochemical method was used to detect the expression of GRINA in different grades and stages of gastric cancer.The correlation between GRINA protein expression and clinicopathological features,prognosis in patients with gastric cancer was compared using the clinical pathological data and follow-up results.4.The lentiviral vector was used to construct stable GRINA knockdown gastric cancer cell lines,and their effects on proliferation,migration and invasion,apoptosis,cell cycle transition of gastric cancer cell lines were detected.5.The effect of GRINA on the proliferation of gastric cancer cells in vivo was investigated by subcutaneous tumor formation in nude mice.6.Bioinformatic analysis of the effect of c-myc on the expression of GRINA at the transcriptional level.7.The results of bioinformatics analysis were verified by chromatin immunoprecipitation assay,and siRNA technology was used to further confirm in the cell lines.8.The expression of Bcl-2 family proteins and cell cycle related regulatory proteins in stably transfected GRINA cells were detected by western blot.Results: 1.The expression of GRINA in cancer tissues was significantly higher than that in normal tissues in the GEO,ONCOMINE,TCGA databases(P < 0.001).2.The expression of GRINA in 20 fresh gastric cancer tissues was significantly higher than that in normal tissues at both mRNA and protein levels.3.Cell Counting Kit-8(CCK8)assay showed that silencing of GRINA significantly inhibit the proliferation ability of gastric cell lines AGS and BGC823.4.Cell proliferation assay(CCK8)results showed that compared with the control group,the proliferation of gastric cancer cell line AGS and BGC823 were significantly inhibited by sliencing of GRINA(P < 0.01).Cell apoptosis assay(Annexin V-FITC/PI staining and flow cytometry analysis)results showed cell apoptosis rate was significantly increasing in gastric cancer cell lines after stable interference of GRINA.5.The wound-healing results showed that silencing of GRINA could significantly inhibit the migration capacity of gastric cancer cells(AGS,BGC823)(P < 0.01)when compared with the control group.Transwell experiments showed that GRINA stable interference cell number was significantly less than the control group,there was significant difference(P < 0.01).7.Compared with the control group,the Bcl-2 family of pro apoptotic protein Bak and Bax were significantly up-regulated,and the expression of anti apoptotic protein Bcl-2 and Bcl-xL protein was significantly reduced in the GRINA silenced cells(P < 0.01).Conclusions: 1.The expression of GRINA in tumor tissues was significantly up-regulated than that in adjacent normal tissues.2.The expression level of GRINA was closely related to the clinicopathological characteristics of gastric cancer,and the expression was negatively correlated with the prognosis of patients with gastric cancer.3.GRINA can promote the proliferation,migration and invasion of gastric cancer cells and inhibit the apoptosis of gastric cancer cells in vitro.4.Stable interference of GRINA could significantly inhibit the ability of subcutaneous tumor formation in nude mice.5.The expression of GRINA in gastric cancer tissues was significantly positively correlated with the expression of c-myc,and its expression was up-regulated by the positive regulation of c-myc protein at the transcriptional level.6.Bioinformatics,chromatin immunoprecipitation assay and cell experiments showed that c-myc could positively regulate the expression of GRINA in gastric cancer cells.6.By regulating the expression of Bcl-2 family proteins and cell cycle related regulatory proteins can GRINA promote the proliferation and inhibit apoptosis in gastric cancer.