| ObjectiveDetecting the expression of Ezrin protein and c-Met protein in normal gastric mucosa,intestinal metaplasia,atypical hyperplasia,gastric cancers and lymph node metastasis immunohistochemically,to analyse the differences and relevance of the two,and to explore the correlation and significance of the expression of Ezrin protein and c-Met protein with gastric carcinogenesis and metastasis.MethodsClinical data and tissue microarray production:Surgically resected specimens of 219 cases of gastric cancer(188 cases at the same time from the edge of greater than 5 cm from the pair of normal gastric mucosa as control),atypical hyperplasia 75 cases, intestinal metaplasia 85 cases and 21 cases of metastatic lymph nodes were collected from Tumor hospilal of Liaoning Province and China Medical University during December 2003 to April 2007.Six blocks of tissue microarray(with tissue sample diameter of 1 mm)including gastric cancer and precancerous lesions,lymph node metastasis were constructed using Microarrayer(United States),4μm consecutive sections were cut,one performed conventional HE staining,others were stored at room temperature for further immunohistochemical staining.Immunohistochemical method and assessment:Envision(PV-9000 two-step) immunohistochemical method was used to detect the expression of Ezrin protein and c-Met protein in normal gastric mucosa,intestinal metaplasia,atypical hyperplasia, gastric cancers and lymph node metastasis.Mouse anti-human Ezrin protein monoclonal antibody(work dilution 1:200)purchased from the company NeoMarkers, Rabbit anti-human c-Met protein polyclonal antibody(ready to use)purchased from Beijing zhong Shan Jinqiao Biological Technology Co.,Ltd.Known positive tissue section was used for positive control,with PBS instead of primary antibodies as a negative control.Immunostaining results assessment:Ezrin protein and c-Met protein positive reaction was defined as clearly brown granules located in the cytoplasm.For each case,200 cells were counted in high-power visual field.Staining intensity of positive cells was assessed into four grades:0,negative;1,light brown;2,brown;3, dark brown.Then stain on the percentage of positive cells:0,no positive cells;1, positive cells≤10%;2,positive cells 11-50%;3,positive cells 51-75%;4,positive cells＞75%.The product of positive staining intensity and percentage of positive cells≥3 were defined as specific immune response,according to the product scores the results were divided into four grades:-(0-2),+(3-4),++(6-8),+++(9-12).ResultsRemoving the shedding of some tissues which was caused by antigen retrieval, finally available samples were 131 cases of normal gastric mucosa,55 cases of intestinal metaplasia,62 cases of atypical hyperplasia,182 cases of gastric cancer and 21 cases with lymph node metastasis.Ezrin protein and c-Met protein located in the cytoplasm.The positive rates of Ezrin protein in intestinal metaplasia(94.55%),atypical hyperplasia(87.10%)and gastric cancer(83.52%)were significantly higher than that in normal gastric mucosa(61.07%)respectively,P＜0.01;In Lauren's types of gastric cancer,the positive rate of Ezrin protein in gastric cancer of intestinal type(93.51%) was significantly higher than that in diffuse type(79.78%),P＜0.01.The positive expression of Ezrin protein in well-differentiated tubular adenocarcinoma(100%)was significantly higher than that in moderately-differentiated tubular adenocarcinoma (91.23%)and poorly-differentiated adenocarcinoma(78.21%),P＜0.01,r_k=0.186.In gastric cancers with lymph node metastasis,the positive rate of Ezrin protein(88.32%) was significantly higher than that in the group without lymph node metastasis(68.89%), P＜0.01;between the primary gastric cancer(71.43%)and relevant lymph node metastases(76.19%),the positive rate of Ezrin protein was no significant difference,P＞0.05.With gender,age,Bormann's and depth of invasion of gastric cancer,the expression of Ezrin protein had no significant correlation.The positive rate of c-Met protein in intestinal metaplasia(63.64%),atypical hyperplasia(61.29%)and gastric cancer(65.93%)were significantly higher than that in normal gastric mucosa(28.24%)respectively,P＜0.01;In Lauren's types of gastric cancer,the positive expressive rate of c-Met protein in gastric cancer of intestinal type (80.52%)was significantly higher than that in diffuse type(59.55%),P＜0.01;the positive expressive rate of c-Met protein in well-differentiated tubular adenocarcinoma (87.50%)was significantly higher than that in moderately-differentiated tubular adenocarcinoma(78.95%)and poorly-differentiated adenocarcinoma(60.26%),P＜0.01,r_k=0.286.In gastric cancer with lymph node metastasis,the positive rate of c-Met protein(71.53%)was significantly higher than that in the group without lymph node metastasis(48.89%),P＜0.05;between the primary gastric cancer(66.67%)and relevant lymph node metastases(74.13%),the positive rate of c-Met protein was no significant difference,P＞0.05.Expression of c-Met protein with patients in sex and age, Bormann's and depth of invasion of gastric cancer had no significant correlation,P＞0.05.The expression of Ezrin protein and c-Met protein in 182 cases of gastric cancer was significantly correlated,r_k=0.602,P＜0.01,and in 21 cases of primary gastric cancer and the relevant lymph node metastases was also closely correlated,r_k were 0.869 and 0.735 respectively,P＜0.01.ConclusionsThe expression of Ezrin protein in intestinal metaplasia,atypical hyperplasia and gastric cancer was significantly higher than that in normal gastric mucosa,which may be involved in the carcinogenesis of gastric mucosa;in intestinal metaplasia and gastric cancer of intestinal type was significantly higher than that in normal gastric mucosa, and the expression of Ezrin protein in the intestinal metaplasia and gastric cancer of intestinal type was no significant difference,suggesting that Ezrin protein may be involved in the occurrence of intestinal metaplasia and gastric cancer of intestinal type. In gastric cancer group with lymph node metastasis,the expression of Ezrin protein was significantly higher than that in the group without lymph node metastasis,which suggested that Ezrin protein may be involved in metastasis of gastric cancer.The expression of c-Met protein in normal gastric mucosa was lower than that in intestinal metaplasia,atypical hyperplasia and gastric cancer;in gastric cancer group with lymph node metastasis was higher than that without metastasis,suggesting that c-Met protein may be involved in the occurrence of gastric cancer and metastasis.The expression of Ezrin protein and c-Met protein in primary gastric carcinoma tissues and the relevant lymph node metastases were significantly correlated,suggested that the two may regulate such as Rho and PI3-K/AKT signal transduction pathways to involve in the occurrence of gastric cancer and lymph node metastasis.
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