Effect Of TGR5 On Malignant Phenotype Of Gastric Cancer Cells

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death in the world.Common risk factors for gastric cancer include Helicobacter pylori,age,tobacco smoking,diet and bile reflux.Bile acids are synthesized from cholesterols in the liver,and are the main components of bile reflux.They play an essential role in the regulation of lipid,glucose and energy metabolism,and also act as signaling molecules.TGR5 is a bile acid receptor localized on the cell membrane,which belongs to the G-protein coupled receptor.Activation of signal transduction is triggered by bile acid when TGR5 coupled with G proteins.Recent studies have shown that TGR5 is abnormally expressed in many types of cancer and may be involved in the development of tumor.Our group have previously confirmed that TGR5 is highly expressed in gastric intestinal metaplasia tissues and promotes expression of CDX2 and MUC2 in gastric epithelial cells through up-regulating HNF4?.Therefore,we examined expression of TGR5 in gastric cancer tissues and explored the function of TGR5 in cells proliferation,migration and invasion of gastric cancer cells,which would provide new experimental evidence to uncover the mechanisms of gastric cancer development.Part 1 Expression and significance of TGR5 in gastric cancer tissuesObjective:This study aimed to explore the expression of TGR5 in GC tissues and analysis the correlation between TGR5 and GC patients'clinical pathological features.Methods:TGR5 was observed in GC tissue microarray containing 105 cases of GC and adjacent tissues using immunohistochemistry.McNemar's?~2 test was used to analyze the expression of TGR5 in different tissues.Pearson?~2 test was used to analyzethe correlation with the clinical pathological features.Kaplan-Meier was used to analyze the relationship between TGR5 expression and GC patients'survival.Results:1.TGR5 localized both on membrane and in cytoplasm of gastric epithelial cells.2.TGR5 expression was presented in both gastric cancer and adjacent tissues,and strong expression of TGR5 was displayed in 43 cases of 105 gastric cancer tissues as well as 10 cases of 105 adjacent tissues(P<0.008).In addition,strong expression of TGR5 was found in 10 of 20 adjacent tissues with intestinal metaplasia as well as 0 of 85 adjacent tissues without intestinal metaplasia(P<0.001).3.In gastric cancer,TGR5 expression was significantly related to tumor size(P=0.018),but not associated with gender,age,pathological grade,TNM stage and distant metastasis.TGR5 expression was not associated with patients'survival.Conclusions:TGR5 expression was higher in gastric cancer tissues compared with adjacent tissues,and the expression level of TGR5 has a significant positive correlation with tumor size,but was not associated with patients'survival.These data suggested a role of TGR5 in development of gastric cancer.Part 2 TGR5 promotes proliferation,migration and invasion of gastric cancer cellsObjective:This study aimed to explore the influence of TGR5 on malignant phenotypes of gastric cancer cells.Methods:qRT-PCR and Western blot was used to detect TGR5 expression in gastric cancer cell lines.MKN-45 and AGS gastric cancer cells were transfected with TGR5 siRNA to knock-down TGR5 expression.Ectopic expression of TGR5 in SGC-7901 cells was achieved by infection with recombinant lentivirus.CCK-8 assay and colony-forming assay were used to evaluate cell proliferation.Flow cytometry was employed to observe cell cycle and apoptosis.Formation of xenografts in nude mice was performed to evaluate in vivo growth.Tanswell assay was used to test cell migration and invasion.The protein levels of?-catenin,Snail and ZEB1 were examined by Western blot.Results:1.Western blot and qRT-PCR revealed that TGR5 was expressed in both GES-1 and gastric cancer cell lines.2.TGR5 promoted cell proliferation.Knock-down of TGR5 in AGS and MKN-45 cells resulted in decreased proliferation,while over-expression of TGR5 in SGC-7901 cells promoted in vitro proliferation.The tumor size,volume and Ki-67 staining intensity of SGC-7901 xenografts in nude mice were increased in LV-TGR5 group compared with control group.3.TGR5 inhibited cell apoptosis but had no effect on cell cycle.Knock-down of TGR5 enhanced apoptosis of AGS and MKN45 cells,whereas over-expression of TGR5 inhibited apoptosis in SGC-7901cells.In AGS,MKN45 and SGC-7901 cells,knock-down or over-expression of TGR5 exhibited no significant influence on cell cycle distribution.4.TGR5 was involved in cell invasion and migration.Transwell assay showed knock-down of TGR5 suppressed cell invasion and migration,while over-expression of TGR5 significantly increased the ability of invasion and migration.Increased expression level of?-catenin,Snail and ZEB1 proteins was exhibited in SGC-7901 cells with ectopic TGR5 expression.Conversely,the level of?-catenin,Snail and ZEB1 protein was decreased in AGS cells with TGR5 knock-down.Conclusions:TGR5 could enhance the proliferation,migration and invasion of gastric cancer cells as well as inhibit cell apoptosis.TGR5 may take a part in metastasis of gastric cancer cells through regulating EMT pathway.