Expression And Clinical Significance Of TGR5 And HNF4α In Gastric Cancer

[Background]Gastric cancer is a common malignant tumor,with the fifth and fourth highest incidence and mortality rates of malignant tumors worldwide.East Asia,including China,is the region with the highest incidence of gastric cancer,and China has the third highest incidence and mortality rate of gastric cancer.The mechanism of gastric cancer has not been fully elucidated,so the study of the process of gastric cancer development is of great significance to understand and prevent gastric cancer.Helicobacter pylori infection is considered to be the most important risk factor for gastric cancer,and eradication of H pylori can significantly reduce the risk of gastric cancer.However,some patients who are not infected with H pylori and those who successfully eradicate H pylori still develop gastric cancer,suggesting the existence of risk factors other than Hpylori infection.Previous studies have reported that patients’ gastric fluid and serum bile acid concentrations are associated with the risk of gastric cancer,suggesting that bile acid stimulation is a risk factor for gastric cancer development.Previous studies have shown that bile acids can directly stimulate gastric mucosal epithelial cell carcinogenesis through their cytotoxicity.However,bile acids can also stimulate gastric carcinogenesis by stimulating cellular bile acid receptors that activate downstream signaling pathways.Bile acid receptors include nuclear receptors and membrane receptors,of which the most important membrane receptor is G protein-coupled bile acid receptor 1(GPBAR1,also known as TGR5),which is expressed in human and rodent tissues,including pancreas,liver,kidney,small intestine,and placenta.Upon activation by bile acids,TGR5 exerts signal transduction through coupled G proteins.It has been found that TGR5 is expressed in normal gastric mucosa,intestinal epithelial metaplasia and gastric cancer tissues,and its expression intensity increases with the severity of gastric mucosal lesions.TGR5 expression correlates with gastric cancer tissue type,and the intensity of TGR5 staining is significantly higher in intestinal gastric cancer than in diffuse gastric cancer.Another study showed that the expression of TGR5 was positively correlated with poor prognosis of gastric cancer patients.Our group found that TGR5 can promote malignant phenotypes such as proliferation,migration and invasion of gastric cancer cells,suggesting that TGR5 molecules are involved in the development of gastric cancer.The development of gastric cancer is often accompanied by abnormal expression of a series of transcription factors,among which Hepatocyte nuclear factor 4a(HNF4α)is considered to be a key transcription factor in gastric carcinogenesis.HNF4α can be regulated by AMPK signaling pathway,and it can also regulate the WNT signaling pathway through its target gene WNT5A,and antagonizing HNF4α with AMPKα agonist metformin has anti-tumor effects.The group has found that in the early stages of gastric cancer prevention and treatment,AMPKα-HNF4α axis has been shown to have anti-tumor effects.Our group found that bile acid could induce HNF4α expression through the activation of TGR5 and ERK1/2 pathways in an in vitro model of gastric mucosal intestinal metaplasia,and high TGR5 levels were associated with high HNF4α levels in intestinal metaplasia tissues.In this study,we analyzed the correlation of the expression level of TGR5 and HNF4α in gastric cancer tissues and different gastric mucosal lesion tissues during gastric cancer progression by bioinformatics analysis and immunohistochemical staining,using paired paracancerous mucosal tissues and tissue samples with chronic gastritis and intestinal metaplasia as controls,respectively;and analyzed the pathological significance of the correlated expression of both,to further explore the role of bile acids in gastric carcinogenesis and development.Part 1Expression and clinical significance of TGR5 and HNF4α in gastric cancer tissues[Objective]The aims of this study were to detect and analyze the expression of TGR5 and HNF4α in gastric cancer tissues,using the paracancerous mucosal tissue as a control.[Methods]The RNA-seq data of gastric cancer cohort in the TCGA database were used to analyze the expression levels of TGR5 and HNF4α in gastric cancer tissues(375 cases),and to analyze the relationship between the expression levels of both and the clinicopathological characteristics and prognosis of gastric cancer patients.Gastric cancer tissues and their paired paracancer mucosal tissues were collected(92 cases),and the expression levels of TGR5 and HNF4α were detected by immunohistochemical staining to verify the conclusions of bioinformatics analysis.[Results]Bioinformatics analysis revealed that there was no statistical difference in the expression level of TGR5 in gastric cancer compared with paraneoplastic tissues(P>0.05);there was a group difference in the expression level of TGR5 in gastric cancer tissues with different TNM stages(P<0.05),which was lower in TNM stage Ⅰ group than TNM stage Ⅱ and TNM stage Ⅲ(P<0.05),and there was no statistical difference in the expression level of TGR5 in the rest of the groups between two comparisons(P>0.05).In samples with different WHO grades of gastric cancer,the mRNA expression level of TGR5 in the GradeⅠ group was lower than that in the Grade Ⅲ group(P<0.05),the mRNA expression level of TGR5 in the Grade Ⅱ group was lower than that in the Grade Ⅲ group(P<0.05),and there was no statistical difference in the expression level of TGR5 between the Grade Ⅰand Grade Ⅱ groups(P<0.05).The median survival of patients in the high TGR5 expression group was shorter than that in the low TGR5 expression group(P<0.05);the expression of HNF4α in gastric cancer was significantly higher than that in paracancerous tissues(P<0.05);the expression levels of HNF4α in different WHO-graded samples were statistically different among the groups P<0.05),and the comparison between the groups showed that The mRNA expression level of HNF4α in Grade Ⅰ group was higher than that in Grade Ⅲ group(P<0.05),and the mRNA expression level of HNF4α in Grade Ⅱ group was higher than that in Grade Ⅲ group(P<0.05),and there was no statistical difference in the expression level of HNF4α between Grade Ⅰ and Grade Ⅱ groups(P>0.05);the median survival time of patients in the group with high expression of HNF4α was higher than that in the group with high expression of HNF4α(P<0.05).The median survival of patients in the group with high HNF4α expression was longer than that in the group with low HNF4α expression(P<0.05).Immunohistochemical results verified that TGR5 expression was upregulated in gastric cancer tissues(P<0.05),and its expression level did not correlate with the clinicopathological characteristics of gastric cancer patients(P>0.05);the expression level of TGR5 in gastric cancer tissues was positively correlated with the poor prognosis of gastric cancer patients(P<0.05);HNF4α expression was upregulated in gastric cancer tissues(P<0.05);its expression level did not correlated with the clinicopathological characteristics of gastric cancer patients(P<0.05).There was no correlation between its expression level and clinicopathological characteristics of gastric cancer patients(P>0.05);The median survival time of gastric cancer patients in the HNF4α high expression group was longer compared with the HNF4α low expression group(P<0.05).[Conclusion]TGR5 expression was up-regulated in gastric cancer tissues,suggesting that TGR5 may be involved in the promotion of bile acid on gastric carcinogenesis and development,and its expression in gastric cancer may receive post-transcriptional regulation;the expression level of TGR5 in gastric cancer tissues was positively correlated with the poor prognosis of gastric cancer patients,suggesting that TGR5 has some predictive value for the prognosis of gastric cancer patients;HNF4α expression was up-regulated in gastric cancer tissues,and HNF4α expression level was negatively correlated with poor prognosis of gastric cancer patients,suggesting that HNF4α may participate in the process of gastric carcinogenesis and inhibit the malignant development of gastric cancer by maintaining the intestinal phenotype of intestinal gastric cancer,and the high expression level of HNF4αmay serve as a predictive marker for good prognosis of gastric cancer patients.Part 2Expression and clinical significance of TGR5 and HNF4α in different gastric mucosal lesion tissues[Objective]The aims of this study were to detect and analyze the expression and clinical significance of TGR5 and HNF4α in chronic gastritis tissues,intestinal metaplasia tissues and gastric cancer tissues.[Methods]Chronic gastritis tissues(60 cases),intestinal metaplasia tissues(62 cases)and gastric cancer tissues(92 cases)were collected,and the expression levels of TGR5 and HNF4αwere detected using immunohistochemical staining.[Results]The expression levels of TGR5 differed in different gastric mucosal lesion tissues(P<0.05),and there was a strong correlation between the expression levels of TGR5 and the degree of gastric mucosal lesions(Cramer’s V=0.573,P<0.05),and its expression levels increased with the progression of gastric mucosal lesions(CG vs IM P<0.05,IM vs GC P<0.05).The expression levels of HNF4α differed in different tissues of gastric mucosal lesions(P<0.05),and there was a strong correlation between the expression levels of HNF4α and the degree of gastric mucosal lesions(Cramer’s V=0.415,P<0.05),and its expression levels differed between chronic gastritis tissues and intestinal metaplasia tissues(CG vs IM P<0.05)and not statistically between intestinal metaplasia tissues and gastric cancer tissues(IM vs GC P<0.05).[Conclusion]The expression levels of TGR5 and HNF4α differed in different gastric mucosal lesions,and their expression levels correlated with the progression of gastric mucosal lesions.The expression levels of HNF4α were not significantly different between the gastric cancer stage and the intestinal metaplasia stage,suggesting that HNF4α plays an important role in promoting the development of intestinal metaplasia.Part 3Correlation of the expression level of TGR5 and HNF4α in gastric mucosal lesions[Objective]The aims of this study were to detect and analyze the correlation between the expression levels of TGR5 and HNF4α in different gastric mucosal lesions and to analyze the clinical significance of their related expression in different stages of gastric carcinogenesis.[Methods]The correlation between the immunohistochemical results of TGR5 and HNF4α in different gastric mucosal lesion tissues was statistically analyzed using the Mantel-Haenszel chi-square test,and the degree of correlation was expressed by Pearson correlation coefficient.According to the expression levels of TGR5 and HNF4α in gastric cancer tissues,gastric cancer patients were grouped as follows:group 1 with low expression of both TGR5 and HNF4α;group 2 with high expression of TGR5 and low expression of HNF4α;group 3 with low expression of TGR5 and high expression of HNF4α;group 4 with high expression of both TGR5 and HNF4α.The differences in 5-year survival rates between the groups were analyzed.[Results]In chronic gastritis tissues,there was a linear relationship between TGR5 and HNF4αimmunohistochemistry score(P<0.05)and a positive correlation between TGR5 and HNF4α expression level(Pearson R=0.288,P<0.05);in intestinal metaplasia tissues there was also a linear relationship between TGR5 and HNF4α immunohistochemistry score(P<0.05)and a positive correlation between TGR5.There was a linear relationship between TGR5 and HNF4α immunohistochemistry score in gastric cancer tissues(P<0.05),and a positive correlation between TGR5 and HNF4α expression level(Pearson R=0.223,P<0.05).Survival analysis showed no statistical difference in 5-year survival rate between the gastric cancer patient groups.[Conclusion]TGR5 and HNF4α were correlated in different gastric mucosal lesion tissues,and TGR5 and HNF4α were jointly involved in the process of gastric carcinogenesis,and there might be a potential regulatory relationship between them.The correlation between TGR5 and HNF4α expression levels in different stages of gastric mucosal carcinogenesis was weaker in chronic gastritis and gastric cancer tissues and stronger in intestinal metaplasia tissues,suggesting the regulatory role of TGR5 and HNF4α in the progression of chronic gastritis to intestinal metaplasia,while the TGR5-HNF4α axis accounted for a decreased share of the molecular regulatory network in the progression of intestinal metaplasia to gastric cancer.The combination of TGR5 and HNF4α did not show better predictive value.