The Study Between TFF1 Polymorphisms And Gastric Cancer Susceptibility And Survival

Gastric cancer(GC)is a complex disease as a result of many factors such as Helicobacter pylori infection,dietary habits and genetic factors.At present,it is the fifth most common cancer and the third leading cause of the cancer-related death in the world with approximately 951,000 new diagnosed cases and 723,000 deaths.More than 70% of the cases worldwide occur in developing countries,and half of the global burden is found in Eastern Asia(mainly in China).In China,according to the epidemiological data,GC is the third major cancer with a high incidence rates(29.24 per 100,000)and ranks the third in cancer mortality.The limited diagnostic capability and therapeutic options result in the poor prognosis with an overall 5-year survival rate around 30% worldwide.The main obstacle is the very little understanding of the mechanisms underlying the development of GC.As known,the pathogenesis of GC is associated with precancerous lesions such as chronic atrophic gastritis.Trefoil Factor 1(TFF1,also named p S2),which is expressed to protect and repair the integrity of mucus layer,belongs to the trefoil factor family peptides that form a clover leaf-like domain through disulfide bonds and are resistant to proteolysis and the acidic environment.TFF1 was first identified in the human breast cancer cell line MCF7,and later was found located in the gastric epithelia and secreted mainly by the upper portion of the pit cells from body to the pyloric sphincter of the stomach.TFF1 is overexpressed in regenerating tissue after the mucosal injury,and lost in about 60% of the gastric tumors.Reduction of TFF1 is also observed in gastric adenomas compared with that in normal adjacent tissues.The deficiency of TFF1,which is important for normal differentiation of antral and pyloric gastric mucosa,may result in the accumulation of undifferentiated gastric epithelial cells that could progress to the malignant cells.According to the previous reports,TFF1 could reduce the cell proliferation by inducing the G1-S transition delay and loss of TFF1 may promote the gastric carcinogenesis cascade by activating the IKK/NF-κB pathway and PP2A-dependent β-catenin signalling.Therefore,TFF1 is considered as a gastric-specific tumor suppressor gene,which was also supported by the strong evidence provided by the tff1-knockout(Tff1-KO)mouse model.The mechanisms of the repression of TFF1 have been studied.The silencing of TFF1 in GC can be explained by loss of heterozygosity(LOH)and promoter hypermethylation.Histone modification such as H3K9 methylation and H3 deacetylation at the tff1 promoter can also induce the absence of tff1 in N-methyl-N-nitrosourea-induced GC mouse model.Moreover,in transcriptional level,the role of CCAAT/enhancer binding protein-β and cofactor of BRCA1 in suppression of TFF1 has been affirmed.However,further investigations are still needed in the mechanisms regulating the TFF1 expression and its role in the gastric tumorigenesis.Genetic alterations may also serve as an important participator in the occurrence of GC.Somatic mutations of the TFF1 gene are only identified in few gastric tumors.Therefore,the studies about the single nucleotide polymorphisms(SNPs),the most common sources of human genetic variations,should be put into our consideration.However,only one study reported the association between single genetic variant in TFF1 promoter and the risk of GC in a very small population of Iranians.No well-designed large sample based association study has been reported.Therefore,we used the tag SNPs to study the relationship between important genetic variations and the complex diseases.In this study,we used two-stage large sample to detect the association of TFF1 polymorphisms with the risk and prognosis of GC.We also used molecular biological experiment to explore the underlying mechanisms.Part Ⅰ.The association between TFF1 polymorphisms and the susceptibility of gastric cancerBackgourd: At present,the mechanisms underlying the regulation of TFF1 were still not clear enough.Genetic alterations may also influence the expression of TFF1.Somatic mutations of the TFF1 gene are only observed in about 5% gastric tumors.The single nucleotide polymorphisms(SNPs)are regarded as the most common sources of human genetic variations.Therefore,it is meaningful to study the TFF1 related SNPs.In present study,it was the first time reporting the association between TFF1 tag SNPs and GC.Methods and Results: We used the Hap Map database to find out the tag SNPs located in the full length of TFF1 gene and its promoter.Finally,5 tag SNPs were identified.By using in a two-step case-control study with large sample size,we analyzed association of these tag SNPs with the risk of GC.TFF1 rs3761376 AA was found associated with increased risk of GC.To further explain the underlying mechanisms,luciferase assays,EMSA and real-time PCR were used.Compared with G allele,rs3761376 A could affect the binding affinity of ER(estrogen receptor)with TFF1 promoter.Conclusion: TFF1 rs3761376 A could reduce the expression of TFF1 by affecting the binding affinity between ER and TFF1 promoter,which finally increased the GC risk.Part Ⅱ.The association between TFF1 polymorphisms and the prognosis of gastric cancerBackgourd: Many studies have reported the association between polymorphisms and the prognosis of cancers.These results indicated the possibility to use SNPs as biomarkers for speculating the survival of patients with caner.So far,no reports about the association between the TFF1 SNPs and the prognosis of GC have been published.Methods and Results: Because two tag SNPs(rs3761376,rs2839488)was observed related with GC susceptibility in the first-stage sample.The second-stage sample contains the data of patients’ survival,so we analyzed the association between the two tag SNPs and the prognosis of GC.The recessive model of rs3761376 indicated the poor prognosis of the patients with lymph nodes metastasis or patients receiving chemotherapy after surgery.The dominant model of rs2839488 was associated with well prognosis of the patients receiving chemotherapy after surgery.By using stepwise Cox regression model,we found the recessive model of rs3761376 and lymph nodes metastasis were identified as the main effect factors associated with the survival of patients who had received chemotherapy after surgery.Conclusion: The recessive model of rs3761376 was related with the poor prognosis of the patients with lymph nodes metastasis or patients receiving chemotherapy after surgery.The dominant model of rs2839488 correlated with well prognosis of the patients receiving chemotherapy after surgery.Moreover,the stepwise Cox regression model indicated the recessive model of rs3761376 and lymph nodes metastasis were the main effect factors associated with the survival of patients receiving chemotherapy after surgery.Part Ⅲ.The effect of TFF1 on 5-FU chemosensitivityBackgourd: According to our previous result,TFF1 rs3761376 AA could affect the binding ability of ER on TFF1 promoter,and reduced the TFF1 expression.And this genotype was also the main effect factor correlated with the prognosis of patients with chemotherapy.Therefore,we hypothesized that TFF1 could influence the chemosensitivity.Methods and Results: First,we overexpressed and repressed TFF1,separately.Then by using CCK-8 and apoptosis assays,we found TFF1 could inhibit cell proliferation and induce apoptosis in the combination of 5-FU.In order to find out the proteins affected by TFF1,we used RNA-sequcening,and found three proliferation or apoptosis related proteins BAD,RBL1 and Pyk2.At last,real-time PCR was used to determine the relation between these proteins and TFF1.Conclusion: TFF1 might enhance the 5-FU cytotoxicty through regulating the expression of BAD,RBL1 and Pyk2.