Biological Function And Mechanism Of CircGLIS2 In Gastric Cancer

Gastric cancer is the common malignant tumor with high occurrence and mortality in the worldwide.With a lack of obvious clinical symptoms at early stage,most cases of gastric cancer have been diagnosed at advanced stage.Despite the development of new diagnostic techniques,surgery,anti-cancer therapy and molecular targeted drugs,the 5-year survival rate of gastric cancer patients is still low.Therefore,it is important for the diagnosis and treatment of gastric cancer to find new tumor markers and therapeutic targets and explore their functions and mechanisms.Circular RNAs（circRNAs）are a class of competing endogenous noncoding RNAs,characterized by their covalently closed-loop structures without a 5’ cap or a 3’ Poly（A）tail.circRNAs have the characteristics of high stability,conservativeness of biological evolution and tissue-specific expression.Current studies have found that circRNAs play an important regulatory role in tumorigenesis and development.So far,there are few studies on the mechanism of circGLIS2 in tumorigenesis and development.This study revealed the relationship between circGLIS2 and the occurrence and development of gastric cancer in vitro and in vivo.The low expression of circGLIS2 in gastric cancer can inhibit the proliferation,migration and invasion of gastric cancer cells.In vitro experiments confirmed the role of circGLIS2/miR-558/MLKL regulatory network in gastric cancer.Dual-luciferase reporter assays and functional recovery experiments confirmed the effects of miR-558 and MLKL on circGLIS2.This indicated that circGLIS2 could affect cell proliferation,migration,and invasion through circGLIS2/mir-558/MLKL regulatory network.The circGLIS2/mir-558/MLKL pathway is a potential target for immunotherapy of gastric cancer.Part I Screening and validation of circRNAs in gastric cancerObjective:To explore the differential expression profile of circRNA in gastric cancer.Methods:Eight cases of gastric cancer and their adjacent tissues were selected to detect the differentially expressed circRNA by circRNA microarray analysis.The changes of 10 selected circRNAs were confirmed by Quantitative real-time PCR（Q-PCR）.Result:A total of 761 downregulated circRNA（fold-change>1.5,P<0.05）were identified in gastric cancer compared with the adjacent normal gastric tissues.The Q-PCR showed that the expression of hsa_circ₀089762（0.431±0.163）,hsa_circ₀036592（0.689±0.110）,circGLIS2（hsa_circ₀005692）（0.631±0.165）and hsa_circ₀000141（0.633±0.171）（P<0.05,respectively）in gastric cancer were significantly lower than that in adjacent normal tissues.Conclusion:The down-regulation of circGLIS2 expression in gastric cancer is negatively correlated with tumorigenesis.Part II Study on the effect of circGLIS2 on proliferation,migration and invasion of gastric cancer in vitroObjective:To investigate the effect of circGLIS2 on proliferation,migration and invasion of gastric cancer in vitroMethods:Construction of lentiviral vector plasmids overexpressing hsa_circ₀089762,hsa_circ₀036592,circGLIS2（hsa_circ₀005692）and hsa_circ₀000141 circRNA,transient BGC-823 cells,Q-PCR to verify the transfection efficiency;Q-PCR to detect of circGLIS2 expression in gastric epithelial cells GES-1 and different gastric cancer cell lines;Construction of gastric cancer cell lines stably expressing circGLIS2 BGC-823 and stably down-regulating circGLIS2 HGC-27;The proliferation of the cells was measured by cell counting kit-8（CCK-8）.The migration ability was analyzed by wound healing assay.The invasive ability was analyzed by Transwell assay.Result:hsa_circ₀089762,hsa_circ₀036592 and circGLIS2 were transfected successfully in BGC-823 cells.The cell viability assay,wound healing assay and Transwell assay showed that over-expression of circGLIS2 could suppress the proliferation,migration and invasion of gastric cancer cell line BGC-823,compared with the control group（P=0.002、0.001、0.44,respectively）.Compared with GES-1,the expressions of circGLIS2 in different gastric cancer cell lines SGC-7901,BGC-823,MGC-803,MKN-45 and HGC-27 were down-regulated,and there were significant differences（P<0.05,respectively）.In HGC-27 cells,circGLIS2 silencing significantly promoted cell proliferation,migration and invasion（P<0.05,respectively）.Conclusion:The expression of circGLIS2 is down-regulated in gastric cancer,and it can inhibit the proliferation,migration and invasion of gastric cancer.Part III Role of circGLIS2/miR-558/MLKL regulatory network in gastric cancerObjective:To study the role of circGLIS2/miR-558/MLKL regulatory network in gastric cancer and its effect on the biological behavior of gastric cancer cells.Methods:Dual luciferase reporter assay was used to validate the specific binding of miRNA with circGLIS2 and the downstream target gene mRNA with miRNA;fluorescence quantitative PCR and Western Blot were used to validate the effect of overexpression or silencing of circGLIS2 on the downstream target gene expression;and functional recovery test was used to further validate the effect of circGLIS2 on the proliferation,migration and invasion of gastric cancer cells.Result:Double luciferase reporter assay confirmed the specific binding of circGLIS2 to microRNA-558 and microRNA-558 to target gene MLKL.Overexpression of circGLIS2 significantly promotes the expression of MLKL gene and protein,which can completely reverse the transfection of microRNA-558 mimic.On the contrary,silencing of circGLIS2 significantly inhibits the expression of MLKL gene and protein,which can completely reverse the transfection of microRNA-558 inhibitor.The effects of circGLIS2/mir-558/MLKL regulatory network on the proliferation,migration and invasion of gastric cancer cells were further verified by the functional recovery experiments of mir-558 and MLKL.Conclusion:circGLIS2/miR-558/MLKL influences the proliferation,migration,and invasion of gastric cancer through competitive endogenous RNA mechanisms.Part IV The effect of circGLIS2 on tumorigenesis of gastric cancer cells in vivoObjective:In vivo experiments confirmed the tumorigenic effect of circGLIS2 on gastric cancer cells.Methods:Balb/c nude mice tumor-bearing models of gastric cancer cells and their control models were constructed and identified.The tumors were measured every 5 days to the 25th day and the tumor volume was calculated following the formula lergth×width2/2.The mice were killed at 25th day after inoculation.Tumor tissues were detected by immunohistochemistry（IHC）.Result:Decreased tumor sizes and weights were observed in the circGLIS2 up-regulation group compared to those in the Lv-NC group.Additionally,increased tumor sizes and weights were observed in circGLIS2 down-regulation group compared to those in the si-NC group.IHC staining showed over-expressed of circGLIS2 up-regulated the expression of MLKL.And silencing of circGLIS2 had the opposite result.Conclusion:circGLIS2 may act as a cancer suppressor gene in gastric cancer cell in vivo.