The Role Of The Membrane Bile Acid Receptor TGR5 In The Pathogenesis Of Non-small Cell Lung Cancer And Its Clinical Significance

ObjectiveThis study is designed to investigate the expression of G protein-coupled bile acid receptor（GPBAR1,TGR5）in non-small cell lung cancer（NSCLC）,and evaluate the pathogenesis and clinical implications.Methods1.Immunohistochemistry（IHC）was used to detect the expression of TGR5 in160 NSCLC tissues and their adjacent lung tissues.The relationship between TGR5expression and clinicopathological features as well as the prognosis of NSCLC patients was analyzed.2.The influence of TGR5 expression on NSCLC cell proliferation,cycle,apoptosis,migration,invasion and tumor growth was detected by means of gene interference or over-expression in vitro and in vivo.Western blot technique was used to detect the effects of TGR5 on the cell cycle regulators,migratory molecules and matrix-degrading enzymes expression so as to clear the molecular mechanisms of TGR5 in regulating the proliferation and metastasis of NSCLC.3.The impacts of TGR5 on Janus kinase2/Signal transduction and transcriptional activation factor3（JAK2/STAT3）signaling pathway were further explored;the transcriptional activity of STAT3 influenced by TGR5 was detected with a luciferase reporter assay;the effects of JAK2/STAT3 pathway on the proliferation,migration and tumor growth in NSCLC cells induced by TGR5 were investigated in vitro and in vivo,and the application of JAK2 inhibitor in the TGR5 over-expressed tumor was discussed.We further detected the expression of phosphorylated STAT3(p-STAT3^Tyr705)in the same NSCLC tissues by immunohistochemistry,then to analyze the correlation between TGR5 and p-STAT3^Tyr705yr705 expression and the survival analysis.4.ELISA method was used to detect the serum levels of bile acids in the NSCLC patients and healthy people.Then we further analyzed the relevance between bile acids contents and TGR5 expression in NSCLC,and clarified the impacts of bile acids on NSCLC cell phenotypes in vitro.Results1.Immunohistochemical results showed that TGR5 expression was significantly increased in NSCLC carcinoma tissues compared with the adjacent tissues（P<0.001）.Patients with higher TGR5 expression had later clinical stage and larger tumor diameter than those with lower TGR5 expression（P<0.05）.TGR5 over-expression was an independent predictor of poor prognosis in NSCLC patients（Hazard ratio[HR]=1.967,95%CI=1.203-3.214,P=0.007）.2.TGR5 promoted cell proliferation in NSCLC by up-regulating the expression of cell cycle proteins such as cyclin D1,cyclin E1,p-RB,c-Myc,CDK4/6 and so on.TGR5 knockdown caused G1 phase arrest in NSCLC cells without affecting cell apoptosis.We further confirmed that TGR5 promoted the growth of transplanted tumor in nude mice in vivo experiments.In addition,TGR5 enhanced the migration and invasion capacity of NSCLC cells by regulating the expression of MMP2,MMP9,RhoA and ROCK1.3.Western blot results showed that TGR5 up-regulated the phosphorylation level of JAK2 and STAT3,and increased the transcriptional activity of STAT3.Additionally,the activation of JAK2/STAT3 pathway mediated the proliferation and migration abilities of NSCLC cells induced by TGR5 over-expression.And the JAK2inhibitor significantly reversed the tumor growth promoted by TGR5 high expression.Moreover,there was a positive correlation between TGR5 and p-STAT3^Tyr705expression in NSCLC tissue samples.Patients with both high TGR5 and p-STAT3^Tyr705yr705 expression had the worst prognosis.4.The serum levels of bile acids in NSCLC patients were significantly higher than those of healthy individuals,such as DCA,CDCA and UDCA.And the level of bile acids was positively correlated with TGR5 expression.Furthermore,DCA promoted NSCLC cell migration by activating TGR5 in vitro.Conclusions1.This study firstly discovered that TGR5 expression was elevated in the tumor tissues of NSCLC patients.The high expression of TGR5 was an independent predictor of poor prognosis in NSCLC patients.2.TGR5 promoted cell proliferation,migration and tumor growth by activating JAK2/STAT3 signaling pathway in NSCLC.3.The serum levels of bile acids in NSCLC patients were higher than those from normal healthy subjects.The bile acids contents were positively correlated with TGR5expression.Bile acids promoted NSCLC cell migration and invasion by activating TGR5.The biological roles of TGR5 in the development of NSCLC were first revealed.Therefore,TGR5 will be expected to be a new target for the prognosis and treatment of NSCLC.