| Gastric cancer is one of the most common malignant tumor in China and even across the world.The latest epidemiological data reveal that gastric cancer ranks fifth of incidence and third of mortality of malignant tumors.The treatment of gastric cancer steps into the new era of biological therapy in recent years from traditional therapy of surgery and radiochemotherapy,with application of molecular targeting drugs and immuno-check points inhibitors.However,the 5-year survival rate of gastric cancer is still below 20%,which mainly due to that the molecular mechanisms underlying gastric cancer development are not fully elucidated.Correa model suggests that gastric cancer,especially the intestinal type of gastric cancer,develops from chronic non-atrophy gastritis to chronic atrophy gastritis,to intestinal metaplasia,to dysplasia,and finally to in situ carcinoma.The risk factors of gastric cancer include infection of helicobacter pylori,high-salt diet,and bile reflux,et al.The relationship of bile reflux and gastric cancer attracted much attention in recent years.Accumulated evidence showed the concentration of bile acids in gastric juice of patients with atrophy gastritis and intestinal metaplasia was much higher than that of non-atrophy gastritis.The follow-up study demonstrated a higher incidence of gastric cancer in patients with higher bile acids in gastric juice than those patients with lower bile acids in gastric juice.We previously found that bile acids could induce expression of mutiple intestinal markers on gastric epithelial cells,and that farnesoid X receptor(FXR)could upregulate SHP,mi R-92 a and mi R-21 and promote bile acids induced intestinal metaplasia.We also found that the expression of FXR was higher in intestinal metaplasia and gastric cancer tissues than that in chronic gastritis.These data suggested a role of FXR in the development of gastric cancer.The present study would explore the influence of FXR on malignant phenotypes of gastric cancer cells and provide new evidence for understanding the role of bile acids and FXR in gastric cancer.Part Ⅰ Expression of FXR in Gastric Cancer Tissues and Cell LinesAims:This study aimed to detect the expression of FXR in gastric cancer tissues and paired para-cancerous tissues and to evaluate the association of FXR with clinicopathological characteristcs of gastric cancer.Methods:1.The expression of FXR in 42 gastric cancer tissues and paired para-cancerous tissues was detemined using immunohistochemical staining and the staining results were semi-quantafied.Pearson χ2 test was used to evluate the association of FXR expression and clinicopathological characteristics,whereas Kaplan-Meier and Log-rank test were used to compare the difference of survival curves.2.Westrern blot and RT-PCR were employed to determine the protein and m RNA expression levels in gastric cancer cell lines AZ-521,BGC-823,MKN-45,SGC-7901 and human immortalized gastric epithelial cell GES-1.Results:1.FXR positive staining was mainly localized in the cell neucleis as indicated by immunohistochemical staining.The high-expression rate of FXR in gastric cancer tissues was significantly lower than that in paired para-cancerous tissues(P<0.05).The high expression rate of FXR in gastric cancer tissues was significantly lower than that in the corresponding adjacent tissues,and the difference was statistically significant(P<0.05).The expression level of FXR in gastric cancer was statistically significant with the TNM stage and tumor differentiation of gastric cancer patients(P<0.05).The five-year survival rate of FXR patients with high expression gastric cancer was significantly better than that of patients with low expression of FXR.2.FXR was universly expressed in GES and all the five gastric cancer cell lines mentioned above,as illustrated by Wesrern-blot and RT-PCR.Among them,gastric cancer BGC-823 cells exhibited the highest level of FXR while gastric cancer MKN-45 cells exhibited the lowest level of FXR.Conclusions:1.The expression level of FXR in gastric cancer tissues was significantly lower than that in paracancerous tissues,and the high expression rate of FXR in gastric cancer tissues was statistically significant with the stage and differentiation of tumor TNM,which was positively correlated with the five-year survival rate of patients,suggesting that FXR may be involved in the development of gastric cancer.2.FXR was expressed in multiple gastric cancer cells,with the highest level in BGC-823 and the lowest level in MKN-45,providing appropriate cell lines for further cell biological study.Part Ⅱ The Regulatory Effects of FXR on Malignant Phenotypes of Gastric Cancer CellsAims:This study aimed to explore the regulatory effects of FXR on proliferation,apoptosis,migration and invasion of gastric cancer cells.Methods:1.The FXR expression was knocked-down in BGC-823 cells with infection of recombinant lentivirus expressing FXR-specific short hairpin RNA(LV-FXR-sh RNA),while FXR was ectopically expressed in MKN-45 cells with infection of recombinant lentivirus expressing FXR(LV-FXR).2.The FXR agaonist,GW4064,was employed to activate FXR in both BGC-823 cells and MKN-45 cells.3.The in vitro proliferation of BGC-823 and MNK-45 cells was evaluated with CCK-8assay.4.The cell cycle distribution and spontaneous apoptosis of BGC-823 cells were analyzed with flow cytometry.5.Transwell assays were used to test the in vitro migration and invasion of BGC-823 and MNK-45 cells.6.The subcutaneous xenografts in nude mice were established to determine the in vivo tumorigenesis ability of BGC-823 and MNK-45 cells.Results:1.The results of Westrern blot and RT-PCR indicated that FXR was successfully knocked-down and overexpressed in BGC-823 and MNK-45 cells,respectively.2.As indicated by CCK-8 assays,1μM of GW4064 had no obvious effects on in vitro proliferation of BGC-823 and MNK-45 cells,whereas 5μM of GW4064 significantly suppressed the proliferation of BGC-823 and MNK-45 cells.3.Knock-down of FXR significantly promoted in vitro proliferation of BGC-823 cells,while overexpression of FXR significantly inhibited in vitro proliferation of MKN-45 cells.GW4064 could suppress the cell proliferation of all the tested gastric cancer cell lines.4.The analysis of cell cycle revealed that knock-down of FXR decreased G0/G1 and increased S phase of BGC-823 cells,and that overexpression of FXR increased G0/G1 and decreased S phase of MKN-45 cells.5.Knock-down of FXR had no effects on spontaneous apoptosis of BGC-823 cells.6.The results of transwell assays demonstrated that knock-down of FXR significantly enhanced in vitro migration and invasion of BGC-823 cells,while overexpression of FXR significantly inhibited in vitro migration and invasion of MKN-45 cells.GW4064 could suppress the migration and invasion of all the tested gastric cancer cell lines.7.Knock-down of FXR significantly promoted in vivo tumorigenesis of BGC-823 cells,while overexpression of FXR significantly inhibited in vivo tumorigenesis of MKN-45 cells.Conclusions:The present study found that knock-down of FXR significantly promoted in vitro proliferation,migration and invasion,increased S phase cell distribution,and enhaced in vivo tumorigenesis of gastric caner cells,while overexpression of FXR and activation of FXR exhibited completely opposite effects on MKN-45 cells.These data suggested that FXR might suppress multiple malignant phenotypes of gastric cancer cells and might be involved in the development of gastric cancer as a negative regulator. |
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