| ObjectiveTo detect the expression of Cancerous inhibitor of protein phosphatase 2A (CIP2A) in normal gastric mucosa, Chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinorma; to analyse the relevance of CIP2A, ki-67 and c-Myc expression in gastric carcinorma; and to explore their relations with gastric tumorigenesis and progression.MethodsClinical data and tissue microarray construction:Surgically resected specimens of 159 cases of gastric carcinoma,97 cases of paired normal gastric mucosa (≥5cm away from the edge of the tumor),45 cases of chronic atrophic gastritis,50 cases of intestinal metaplasia and 28 cases of dysplasia were collected from No.l Hospital of China Medical University. Tissue microarrays including gastric carcinomas and precancerous lesions were constructed using Microarrayer,4μm consecutive sections were cut, one performed conventional HE statining, others were stored at room temperature for further immunohistochemical staining.23 cases of gastric carcinoma tissue and corresponding normal gastric mucosa were analyzed by western blot for CIP2A expression.Immunohistochemical method and assessment:PV9000 method was used to detect the expression of CIP2A, Ki-67 and c-Myc in normal gastric mucosa, intestinal metaplasia,dysplasia and gastric carcinoma. Rabbit polyclonal antibody against human CIP2A (working dilution 1:180) was purchased from NOVUS BIOLOGICAL Company. Mouse monoclonal antibodies against human c-Myc (ready to use) and ki-67 (ready to use) were from Fuzhou Maixin Company (China). For negative controls, sections were treated with 0.01 mol/L phosphate-buffered saline instead of primary antibodies. Immunohistochemical staining results assement:CIP2A was located in the cytoplasm and membrane of carcinoma cells; c-Myc was located in the cytoplasm and nucleus of carcinoma cells. Ki-67 was specifically located in the nucleus of proliferating cells. 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) that accounted in 200 cells were divided into 0 (＜5%),1 (5%-25%),2 (26%-50%),3 (51%-75%) and 4 (＞75%). According to the results of the product of A and B, the immunostaining result was classified as 0, negative (-); 1-4, weakly positive (+); 5-8, moderately positive (++) and 9-12, strongly positive (+++).Western blotting for CIP2A: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 100 V for 2 hours, and the membrane was incubated with primary antibody (CIP2A,1:280) 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.RusultsThe positive rates of CIP2A presence in Chronic atrophic gastritis (36/45,80%), intestinal metaplasia (46/50,92%), dysplasia (17/28,60.7%) and gastric cancer (121/159,76.1%) were significantly higher than that in normal gastric mucosa (33/97, 34.02%), P＜0.05; There was no statistical difference between CIP2A expressions in the dysplasia and gastric cancer, P＞0.05; The expressive level decreased from well-differentiated adenocarcinoma (8/9,88.9%) through moderately-differentiated adenocarcinoma (51/59,86.4%) to poorly-differentiated adenocarcinoma (42/66, 63.6%), with significant Rank correlation, rk=-0.317, P＜0.001. In Lauren's types of gastric carcinoma, the positive rate of CIP2A in intestinal gastric carcinoma (61/70, 87.1%) was significantly higher than that in diffused gastric carcinoma (46/73,63%), P =0.001. There was no significant correlation of the expression of CIP2A with patients' gender, age, Borrmann's classification of gastric carcinoma, P＞0.05; In our western blot analysis the expression level of CIP2A was higher than that in normal gastric mucosa (88.49±2.88 vs.79.63±2.52,t=2.314 P=0.025).The positive rates of c-Myc in intestinal metaplasia (46/50,92%) and gastric carcinoma (130/159,81.8%) were significantly higher than that in normal gastric mucosa (62/97,63.9%), P＜0.05; the expression level decreased from well-differentiated adenocarcinoma (8/9,88.9%) through moderately-differentiated adenocarcinoma (51/59,86.4%) to poorly-differentiated adenocarcinoma (51/66,77.3%), with significant Rank correlation, rk=-0.254, P=0.003. In Lauren's types of gastric carcinoma, the positive rate of c-Myc in intestinal gastric carcinoma (60/70,86.5%) was higher than that in diffused gastric carcinoma (55/73,73.9%), but there was no statistical significance, P＜0.05. There was no significant correlation of the expression of c-Myc with patients' gender, age, Borrmann's classification of gastric carcinoma, P＞0.05.The positive rates of Ki-67 in intestinal metaplasia (41/50,82%) and gastric carcinoma (105/159,66%) were significantly higher than that in normal gastric mucosa (47/97,48.5%), P＜0.05; In Lauren's types of gastric carcinoma, the positive rate of c-Myc in intestinal gastric carcinoma (51/70,72.9%) was higher than that in diffused gastric carcinoma (43/73,58.9%), but there was no statistical significance, P＜0.05. There was no significant correlation of the expression of c-Myc with patients' gender, age, Borrmann's classification of gastric carcinoma,P＞0.05. In 159 cases of gastric carcinoma, the expression of CIP2A and Ki-67 was significantly positively correlated, rk=0.420, P＜0.01; The expression of CIP2A and c-Myc in 159 cases of gastric cancer was significantly correlated, rk=0.442, P＜0.01.ConclusionsThe positive rates of CIP2A presence in Chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric cancer were significantly higher than that in normal gastric mucosa, suggesting that CIP2A may play an important role as a carcinogenic factor involved in the carcinogenesis of gastric mucosa; The expression level decreased from well-differentiated adenocarcinoma through moderately-differentiated adenocarcinoma to poorly-differentiated adenocarcinoma, with significant Rank correlation, suggesting that CIP2A expression was correlated with histologic differentiation of gastric carcinoma cells. CIP2A was overexpressed in intestinal metaplasia and in intestinal gastric carcinoma, indicating that CIP2A may be involved in the carcinogenesis of intestinal type gastric carcinoma.The positive rates of c-Myc Ki-67 and Ki-67 in intestinal metaplasia and gastric carcinoma were significantly higher than that in normal gastric mucosa, In Lauren's types of gastric carcinoma, the positive rate of c-Myc and Ki-67 in intestinal gastric carcinoma was higher than that in diffused gastric carcinoma, but there was no statistical significance. c-Myc and Ki-67 protein are biomarkers for proliferating cells, which can be used as a marker of proliferative activity in gastric cancer.The expression of CIP2A and Ki-67 in 159 cases of gastric carcinoma was significantly correlated, suggesting that CIP2A may play an important role in promoting cell proliferation and cell cell malignant transformation. The expression of CIP2A and c-Myc was significantly correlated, which provided a new approach to reduce the expression of c-Myc in gastric cancer, CIP2A might serve as a new therapy target of gastric cancer, which needs to be further researched. |
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