| ObjectiveTo detect the expression of Cripto-1 andβ-catenin protein in normal gastric mucosa, chronic atrophic gastritis (CAG), intestinal metaplasia (IM), dysplasia and gastric carcinoma, to analyse the differences and relevance of their expressions, and to explore their relations with gastric carcinogensis and metastasis.MethodsClinical data and tisue microarry construction:Surgically resected specimens of 192 cases of gastric carcinoma (117 case at the same time from the edge of greater than 5cm from the of normal gaetric mucosa as control),42 cases of chronic atrophic gastritis,48 cases of intestinal metaplasia, and 25 cases of dysplasia were collected from N0.1 Hospital of China Medical University. Tissue microarrys including gastric carciomas and precancerous lesions were constructed using Microarrayer,4μm consecutive sections were cut, one performed conventional HE staining, others were stored at room temperature for further immunohistochemical staining.32 gastric carcinoma specimens and matched normal tissues were detected by western blottingImmunohistochemical method and assessment:The PV-9000 immunohistochemical (IHC) method was used to detect the expression of Cripto-1 andβ-catenin in normal gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma. The PV-9000 kit was purchased from Beijing Zhongshan Golden Bridge Biotechnology Company. Mouse monoclonal antibody against human CR-1 was from the R&D systems (working dilution 1:80). Rabbit polyclonal antibody against human β-catenin was from the Wuhan Boster Biotechnology Company (working dilution 1:50). All procedures were implemented according to the manufacturer's instructions. For negative controls, sections were treated with 0.01 mol/L phosphate-buffered saline instead of primary antibodies.Immunohistochemical staining evaluation:Specific immunoreactivity of CR-1 andβ-catenin protein was located in the cytoplasm. Two hundred cells from two selected representative fields of each section were counted by two independent observers for the determination of their immunostaining intensity. Staining intensity (A) was classified as 0 (negative),1 (weak),2 (moderate) and 3 (strong). The percentage of positive cells (B) examined in 200 cells were divided into 0 (< 5%),1 (5%-25%),2 (26%-50%),3 (51%-75%) and 4 (> 75%). According to the product of A and B, the IHC result was classified as 0, negative (-); 1-4, weakly positive (+); 5-8, moderately positive (++) and 9-12, strongly positive (+++).Western blotting for CR-1 andβ-catenin:Lysis buffer solution was used to extract protein from tissues. Protein concentration was determined by Coomassie brilliant blue, and 10%SDS polyacrylamide gel electrophoresis was performed under 120V. The protein was transferred to nitrocellulose membrane under 100V for 2 hours, and the membrane was incubated with primary antibody (CR-11:300 andβ-catenin 1:500) in 4℃over night, which was further incubated with secondary antibody (1:10000) in room temperature for 2 hours. Immunological straps were finally developed with immunoblotting chemoluminescene reagent (ECL reagent). The electrophoretic results were scanned into images. Data were collected by QUANTITY ONE 4.6 software and the ratio of strip density toβ-actin density was used in statistical analysis.ResultsThe results of immunohistochemistry show that the positive rates of CR-1 expression were significantly higher in CAG (69.0%), IM (83.3%), dysplasia (80.0%) and GC (72.4%) than in normal gastric mucosa (47.9%, P＜0.05), respectively. In Lauren's types of gastric cancer, the positive rate of CR-1 in gastric cancer of intestinal tyre(84.0%) was significantly higher than that in diffuse type(67.4%), P＜.05. The positive rate of CR-1 in moderately-differentiated tubular adencarcinoma(85.7%)was significantly higher than that in poorly-differentiated adencarcinoma(63.5%), P＜.O1. In GC with lymph node metastasis, the positive rates of CR-1 (76.6%) was significantly higher than those without metastasis (60.8%), P＜0.05. The expression of CR-lprotein was not correlatied with age, gender and Borrmann's classification of GC. The increase of CR-1 expression in GC is also demonstrated in our western blot analysis (95.19±29.68 vs78.04±29.02),t=2.338, P=0.023.The results of immunohistochemistry show that the positive rates ofβ-catenin expression were significantly higher in CAG (63.0%), IM (72.2%), dysplasia (64.0%) and GC (57.3%) than in normal gastric mucosa (30.9%, P<0.01), respectively. In IM, the positivity rates ofβ-catenin (72.2%) was significantly higher than in GC (57.3%, P＜0.05). In Lauren's types of gastric cancer, the positive rate ofβ-catenin in gastric cancer of intestinal type(64.6%) was higher than that in diffuse type(51.7%) but with no statistical significance, P=0.07. In GC with lymph node metastasis, the positive rates ofβ-catenin (64.8%) was significantly higher than those without metastasis (39.2%), P<0.05, but also not related to the age and gender of patients, or Borrmann's classification of GC. No statistically significant difference between P-catenin expression in gastric carcinomas (115.97±27.521) and normal gastric mucosa (105.95±23.353) was found in our western blot analysis. t=1.571, P=0.122.In 192 cases of gastric carcinoma, the expression of CR-1 andβ-catenin protein was significantly positively correlated, rk=0.220, P<0.001.ConclusionsThe positive rates of CR-1 and P-catenin expression were significantly higher in gastric cancer and precancerous lesions than in normal gastric mucosa, indicating that they may play important roles as tumorigenic factors and early gastric tumorigenic molecules during gastric carcinogenesis. Our results showed a positive correlation between CR-1 andβ-catenin expression and lymph node metastasis, indicating that they may participate in the development and lymph node metastasis of GC. CR-1 and P-catenin can act as a prognostic indicator for GC patients, but the relevant molecular mechanism requires further investigation.Their expressios in GC was positively correlated, indicating that they are closely related in affecting gastric carcinogenesis, but the relevant molecular mechanism requires further investigation.
|
PDF Full Text Request