| Objective: Gastric cancer(GC)is one of the most common causes of cancer in the world,and gastric cancer ranked the second in the world of cancer related death.In recent decades,the number of esophagogastric junction cancer was significantly increased,and the incidence rate of tumor in gastric distal area was significantly decreased.In the high incidence area of esophageal and gastric cancer in Hebei Province,gastric cardia adenocarcinoma(GCA)also increased markedly.Generally,gastric mucosa will be repaired quickly because of the protective factors,when it was in damage.However,the decrease or loss of protective factors would lead gastric mucosa to be involed in harmful substances,which may affect the expression and fuction of tumor associated gene,even resulted in gastric cancer.Gastrokines(GKN),secreted in gastric mucosa,could inhibit cell growth in gastric cancer.It was reported that Hp infection may reduced the expression of GKN1 and GKN2,indicating that GKNs have closed relationships with development of gastric cancer.Trefoil factor family(TFF),including TFF1,TFF2 and TFF3,are also secreted by gastric mucous to the mucosal surface.Meanwhile,TFFs expressions are associated with MUC secretion.TFFs could induce epithelial cells to move,rapidly repair,and prevent the occurrence of inflammation.Furthermore,TFFs might regulate cell differentiation in gastric mucosa,and are closely related with gastric cancer.Previous studies have found that Hp infection can affect the expressions of TFFs and GKN in gastric epithelial cells.It was reported that Hp infection need to be combined with TFF1,which may be the receptor of Hp.Meanwhile,TFF1 may be combined with GKN2 to form a complex to protect the normal gastric mucosa.The dissociation of TFF1 and GKN2 in cells could make two proteins lose their normal function,thus promoting the development of tumor.However,the roles of TFFs and GKN2,and relationship between Hp infection and protective factors in the development of gastric cancer at different sites are still unclear.Therefore,the expressions of TFF1,TFF2 and GKN2 will be detected and compared between adenocarcinomas of gastric cardia and distal stomach using IHC.Meanwhile,cell growth and expressiongs of profective factors will be studied in GES-1 with Hp Cag A treatment in vitro.Our studies will provide a scientific theoretical basis for the reasonable prevention and control of gastric cancer.Methods: One hundred and thirteen patients with gastric cardiac and distal adenocarcinomas consecutively operated at the second hospital of Hebei Medical Universty in Hebei Province from January 2011 to December 2014,were included in this study.All patients were underwent a preoperative upper gastrointestinal endoscopy(with biopsy specimens routinely performed).Sixty-one patients with cardiac carcinoma attended in this study were chosen according to the tumor center within 1 cm above and 2 cm below the anatomic esophagogastric junction(EGJ).Fifty-two cases with the tumor at antrum were operated with different surgical approaches.Twenty-three cases with normal gastric epithelial mucosa were also collected as control group in the study.The expressions of TFF1,TFF2 and GKN2 were detected by immunohistochemistry method between between gastric cardia and distal adenocarcinomas.Furthermore,the expressions of TFF1,TFF2 and GKN2 and cell proliferation were also studied in GES-1 using cell culture,Real-time PCR and Western Blot in vitro.Results:1 Expression of TFF1,TFF2 and GKN2 in gastric cancer The positive staining of TFF1,TFF2 and GKN2 showed brown granules in cytoplasm.The positive rate of TFF1,TFF2 and GKN2 in gastric cancer was 42.5%(48/113),46.9%(53/113)and 29.2%(33/113),significantly lower than that in normal gastric mucosa,respectively(P<0.05).2 The expression of TFF1,TFF2 and GKN2 in GCA and DGA2.1 Expression of TFF1 in GCA and DGA IHC results showed that the positive rate of TFF1 was significantly lower in GCA than in NGM(42.6%vs87.0%,P<0.05).The expression of TFF1 in DGA(42.3%)showed markedly lower than in NGM(P<0.05).However,there was no significant difference between GCA and DGA.In DGA,the expression of TFF1 was closely related to invasion depth and lymph node metastasis(P<0.05).In contrast,TFF1 expression had no distinct associationwith clinicopathological features in GCA.2.2 Expression of TFF2 in GCA and DGA The positive rate of TFF2 expression was 49.2%,44.2% in GCA and GDA respectively,significantly lower than that in normal gastric mucosa(91.3%,P<0.05).The comparative study showed no difference in TFF2 expression between GCA and DGA(P>0.05).In GCA and DGA,the expression of TFF2 was not related with gender,age,invasion depth,lymph node metastasis and tumor stage(P>0.05).2.3 GKN2 expression in GCA and DGA IHC results showed that the positive rate of GKN2 in GCA and DGA were29.5% and 29.5%,significantly lower than in NGM(77.3%,P<0.05).There was no significant difference about GKN2 expression between GCA and DGA.In DGA,there was closely relationship between GKN2 expression and lymph node metastasis(P<0.05).However,the expression of GKN2 was not related with lymph node metastasis in GCA(P<0.05).3 The effect of Hp infection on TFF1,TFF2 and GKN2 in GCA and DGA In 113 cases,there were 61 cases of GCA,including 45 cases with Hp infecton and 16 cases without Hp infection.IHC showed that the positive rate of TFF1 expression was 33.3% in samples with Hp infection,lower than that in ones without Hp infection(68.8%,P<0.05).The positive rate of TFF2 in cases with Hp infection was also significantly lower than without Hp infection (40.0% vs 75.0%,P<0.05).GKN2 expression also showed lower sequence in cases with Hp infection.However,there was no difference on TFF2 expression between cases with and without Hp infection.Forty cases were infected by Hp in 52 cases of DGA.The expressions of TFF1,TFF2 and GKN2 in samples with Hp infection were 32.5%,35.0% and20.0% respectively,significantly lower than that in cases without Hp infection(P<0.05)4 The effect of Hp Cag A on cell proliferation in GES-14.1 Cell growth in GES-1 treated with Cag A MTT results showed that the cell survival rates in Cag A treatment groups displayed a dose dependent manner.At 10?g/ml concentration,the cells reached the highest number(P<0.05).Then,the cell survival rates of GES-1gradually decreased with dose increase.4.2 The expression of CDK4 ? Cyclin B1 ? PCNA in GES-1 after Cag A treatment Cag A treatments for 24 h with 2.5?g/ml,5?g/ml and 10?g/ml concentration could significantly increase the expression of CDK4,Cyclin B1 and PCNA at m RNA level(P<0.05).Compared to the control group,Cag A also significantly increased the protein expressions of the three antigens(P<0.05).5 The effect of Cag A on expressions of TFF1,TFF2 and GKN2 in GES-15.1 The expressions of TFF1,TFF2 and GKN2 at m RNA level Real-time PCR results showed that the expression of TFF1,TFF2 and GKN2 in 10?g/ml groups were significantly higher than other groups after 3h treatment(P<0.05).However,after 6h,the m RNA levels of TFF1,TFF2 and GKN2 in 10?g/ml group were significantly lower than the control group(P<0.05).At 24 h,the expressions of TFF1,TFF2 and GKN2 in 2.5?g/ml Cag A groups were significantly lower than that in control group(P<0.05).It indicated that different concentration or treated time of Cag A could affect the m RNA expression of these protective factors,and then decrease the protective function.5.2 The protein expressions of TFF1,TFF2 and GKN2 in GES-1Western blot results showed significantly lower expressions of TFF1,TFF2 and GKN2 in GES-1 after Cag A treatment for 24 h than control group,respectively.Conclusions:1 The expressions of TFF1,TFF2 and GKN2 in GCA and DGA were significantly lower than that in normal gastric mucosa.However,there was no significant difference about three antigens expression between two groups.It suggested that the deficiency of protective factors may play a crucial role in the incidence of gastric cancer,but not obviously related to gastric subsites.2 Hp Cag A could significantly inhibit the expression of TFF1,TFF2 and GKN2 in GES-1 cells,suggesting that Hp infection may reduce the secretion of gastric mucosal protective factors,increase gastric mucosal injury,and then might lead to gastric cancer.3 Cag A treatments with lower concentration could increase cell growth and the expression of CDK4,Cyclin B1 and PCNA in GES-1,which suggested that Hp infection may affect proliferation activity of gastric mucosal epithelial cells,resulting in cell damage. |
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