Long Non-coding RNA DGCR9 Enhances Gastric Cancer Cell Proliferation And Migration Through Glucose Metabolism

ObjectiveWith the development of the diagnosis and treatment in gastric cancer,the incidence of gastric cancer had shown a decreasing trend.However,the incidence of advanced gastric cancer in China was steadily increasing.In this study,we first screened the dysregulated expressed IncRNAs in gastric cancer associated-lncRNA microarray database.The high expression of IncRNAs were selected in gastric cancer tissues,and the target IncRNA which expressed large variations was detected.Although IncRNA had been widely studied in the development of tumor,its biological function in the progress of gastric cancer was not clear.We had analyzed the expression level of the target IncRNA in gastric cancer tissues and the biological role of the target IncRNA in gastric cancer cells.Consequently,the aim of this study was to explore the expression of target IncRNA in gastric cancer tissue and its function of gastric cancer cells.Methods1.Formalin-fixed,paraffin embedded(FFPE)gastric cancer tissues and adjacent non-cancer tissues(NAT)were obtained from 120 patients who underwent primary gastric cancer resection between November,2016 and January,2017.Finally,102 patients with gastric cancer tissue were selected according to this research requirements.2.Based on the information analysis of gene length,chromosome localization and fold change in gastric cancer associated-lncRNA microarray database,the target IncRNA in gastric cancer tissues and adjacent non-cancer tissues was screened.The expression level of the IncRNA in gastric cancer tissues and adjacent non-cancer tissues was detected by qRT-PCR method,and its relationship with clinicopathological factors was analyzed.3.The human gastric epithelial cell line(GES-1)and human gastric cancer cell lines(AGS,HGC-27,MGC-803 and SGC-7901)were cultured.The expression level of target IncRNA in gastric cell lines were detected by qRT-PCR,and the expression of the IncRNA in gastric cancer tissues and gastric cancer cell lines was compared.4.Through liposome by transient transfection,chemically modified small interfering RNA(siRNA)was introduced into gastric cancer cell lines(AGS and MGC-803),to suppress the expression level of the target lncRNA.With the reduction of IncRNA expression level,we used CCK8 assay,colony formation,wound healing,Transwell and glucose uptake assay to demonstrate cellular proliferation,migration and glucose metabolism of gastric cancer cells.5.With lentivirus in stable transfection,AGS and MGC803 gastric cancer cells were transfected with a overexpressing lentivirus expressing the target lncRNA,and the expression level was judged by qRT-PCR.In the end,we used the same assays to verify cellular proliferation,migration and glucose metabolism in gastric cancer cells.Results1.To study the roles of lncRNAs in gastric cancer,we first screened the high expressed lncRNAs in gastric associated-lncRNA microarray database.Then we used GO/Pathway analysis to further clarify the possible functions and mechanisms of the IncRNAs.Finally,DiGeorge syndrome critical region gene 9(DGCR9)was selected as the candidate.2.DGCR9 expression was significantly increased in gastric cancer tissues compared to NAT.DGCR9 also showed a higher expression in gastric cancer cell lines(HGC27,AGS,MGC803,and SGC7901)than that in the normal gastric epithelial cell line GES-1.3.Receiver operating characteristic(ROC)curve analysis was used to evaluate the potential gastric cancer by using DGCR9.DGCR9 showed potential predictive value with an area under curve(AUC)of 0.691.4.To assess associations among DGCR9 expression and clinicopathological characteristics of patients with gastric cancer,patients were divided into two groups,a high expression group(fold-change>2)and a low expression group(fold-change<2).DGCR9 was significantly associated with lymph node invasion(P = 0.011)and TNM stage(P = 0.018).5.In functional deficiency study,a significant reduction in DGCR9 expression by Si-DGCR9 was observed by qRT-PCR.Cell proliferation,cell migration and glucose uptake of AGS and MGC803 cells were reduced with Si-DGCR9 when compared to cells treated with Si-NC.6.AGS and MGC803 gastric cancer cells were transfected with a lentivirus expressing DGCR9(LV-DGCR9),and as judged by qRT-PCR,the expression of DGCR9 was remarkably up-regulated when compared to cells transfected with a negative control lentivirus,LV-NC.Similarly,cell proliferation and cell migration,as well as glucose uptake were significantly promoted by LV-DGCR9 when compared to LV-NC.ConclusionsDGCR9 was significantly expressed in gastric cancer tissues and cell lines.Dysregulated expression of DGCR9 might lead to the carcinogenesis,and was associated with lymph node invasion and TNM stage.Furthermore,knockdown of DGCR9 suppressed cell proliferation,migration,and glucose uptake with opposite effects upon over expression.Overall,DGCR9 may represent a novel biomarker for the diagnosis of gastric cancer.