Expression Of Protein 4.1 In Gastric Carcinoma Tissues And Screen Absent Cell Lines

BACKGROUNDProtein 4.1, which is named after its location on erythrocyte membrane proteins by SDS-PAGE, is basic ingredient of cell membrane skeleton based on spectrin protein in red blood cells. It connects spectrin with actin and it is very important in maintaining normal shape and biophysical characteristics of cells as well as in regulating cell caryomitosis. The absence of protein 4.1 gene will lead to deformity of red blood cells and haemolysis. Protein 4.1 family includes 4.1R (mainly expresses in erythrocyte), 4.1N (neuron-specific expression), 4.1G (broad expression) and 4.1B (expresses mainly in brain), these four members have three same domains: SABD (spectrin-actin-binding domain); FERM domain (4.1-ezrin-radixin-moesin domain) or membrane binding domain (MBD) and CTD (C-terminal domain).Gastric cancer is alimentary tract malignant tumor which hazards human's health heavily. In China, according to the statistics of Ministry of Health, gastric cancer ranks the first in the early 1990s cancer mortality. It is of great clinical and social value to deeply investigate the pathogenesis and the methods of early diagnosis and prognosis assessment of gastric cancer.Some researches revealed that in meningioma, ependymoma, non-small cell lung cancer, breast cancer and other tumors, the expression of protein 4.1 was absent, and an increasing number of clinical and experimental evidences confirmed that the genes of protein 4.1 displayed the role of anti-oncogene in a variety of tumors. So far, the relation between family members of protein 4.1 and the incidence of gastric cancer has not yet been reported. Taking into account that the sequence of family members of protein 4.1 is of high homology, their functions may also have similarities. In this study, we investigated the relation between the expression of family members of protein 4.1 in gastric cancer and the pathogenesis of gastric cancer.Immunohistochemical staining technique is a commonly used technology to detect protein in tissues or cells. It has been extensively used in morphological field. Many new immunohistochemical techniques emerged such as SP, ABC, SABC and Envision~+ methods. Some researches showed that compared with other methods, Envision~+ method had higher sensitivity, and could reduce the hyper-staining and nonspecific staining of organizations. Envision~+ method connected polymerizer with secondary-antibody and enzyme, while there doesn't contain such polymerizer in human tissues, so it will avoid the biotin interference of human tissues and will have more satisfactory results. Because of this, Envision~+ method was used to detect the expression of family members of protein 4.1 in the gastric cancer tissues in our research.PURPOSE1. Envision~+ immunohistochemical staining method was used to detect the expression of family members of protein 4.1 (4.1B, 4.1R, 4.1G and 4.1N) in 104 cases of gastric cancer and 68 cases of normal tissues. We analysed the relation between the expression of protein 4.1s and clinical pathology, and explored the correlation between their expression and gastric cancer, with a view to provide basis for diagnosis, treatment and prognosis in the gastric cancer.2. Screen 4.1 absent cell line in order to provide materials to investigate the function of tumor suppression of protein 4.1.METHODS1 Immunohistochemistry of gastric carcinoma tissues1.1 Obtaining, classification and section of samples Surgical samples of gastric cancer were collected from January of 2005 to June of 2006. Among a total of 104 cases includes 77 cases of male, 27 female, the median age of 61.7 (35-88) years. Each case had the clinical data and surgical records in detail. All cases were not treated by radiotherapy, chemotherapy and immunotherapy before surgery. There were 51 cases which with lymph node metastasis. Refered to the differentiation classification criteria in 1981 recommended by WHO: 30 cases with well-differentiated adenocarcinoma, 28 cases with differentiated adenocarcinoma and 46 cases with poorly differentiated adenocarcinoma. Meanwhile 68 cases of normal gastric mucosa which corresponded with cancer control-standard were obtained from the 104 cases and they were treated as a control group. Samples have been fixed by 4% formaldehyde solution, dehydrated by gradient alcohol, transparent, dipped in wax and embedded by paraffin. Before experiment paraffin block is sliced to 3—4μm/ unit.1.2 HE (hematoxylin - eosin) stainingHE staining was used to identify pathological characteristics of the experimental samples with the original hospital pathological data.1.3 Envision~+ immunohistochemistry of experimental samplesThe expression of 4.1B, 4.1R, 4.1G and 4.1N were detected in the experimental group and the control group simultaneously. Concrete steps: The units were baked at 65°C for 3h, gradually dewaxing and hydrating. Then operate in accordance with the Envision~+ immunohistochemical staining kit specification of Dako Corporation. Note: Different antibodies corresponding different antigen retrieval methods: 4.1NHP with a high pH antigen retrieval solution, thermal repair for 20min; 4.1BU2 with a low pH antigen retrieval solution, thermal repair for 20min; 4.1RE13 with a high pH antigen retrieval solution, thermal repair for 20min; 4.1GU3 with the protease-restored, at room temperature for 5min. The vessels in tumor tissues as a positive control; PBS is used to replace first-antibody for the negative control.1.4 Immunohistochemical determination of the resultsIn munohistochemical staining, the positive result was brown particles which stained cytoplasmic membrane or intracytoplasm. The results were judged by two pathological doctors blindly. The results were assessed by semi-quantitative score standards, the standards were as follows: under high-power microscope, none color recorded as 0, light brown recorded as 1, brown recorded as 2, dark brown recorded as 3. In the same tissue section, because of different differentiation, there were also differences in staining. In such situation, points were recorded by dominant staining; positive cells≤10% recorded as 0, 10-50% recorded as 1, 50-75% recorded as 2, more than 75% recorded as 3. The two part added was the total staining score. 0-1 is"-", 2 is weak positive "+", 3-4 is positive"+ +", 5-6 is strong positive "+ + +".1.5 Analysis of experimental resultsSAS 9.13 was used to do statistical treatment. Chi-square test was used to analyze the relation between the expressions of 4.1B, 4.1R, 4.1G and 4.1N and the clinical pathology.2 Screen 4.1 absent cell lineTumor cell lines EC109, EC9706, EC1 were cultured and detected the expression of protein 4.1s with immuocytochemistry and Western blot.RESULTS1. HE-staining results confirmed the pathology and differentiation types assessed by clinical diagnosis.2. The expression of 4.1N, 4.IB, 4.1R and 4.1G in Gastric cancers and normal tissues: the expressed levels of 4.1B, 4.1R, 4.1G and 4.1N in gastric cancer tissues were significantly lower than the control group (P <0.01).3. The relation between the expression of 4.1B, 4.1R, 4.1G and 4.1N and the differentiation: the expression of 4.1B, 4.1G and 4.1N reduced when the differentiation of cancer tissues became low (P _B <0.05, P _g, P _n <0.01), the expression level of 4.1R had a weakening trend following with the degree of tumor differentiation reduction. However, the difference was not statistically significant (P_r >0.05).4. The relation between the expression of 4.1B, 4.1R, 4.1G, 4.1N and lymph node metastasis: the expression level of 4.1N and 4.1B with lymph node metastasis did not have statistically significant differences (P _B<0.05, P _n< 0.01). The expression of 4.1N and 4.1R associated with lymph node metastasis had no statistically significant differences (P _N, P_r >0.05).5. The relation between the expression of 4.1B, 4.1R, 4.1G, 4.1N and the depth of invasion: Because there were not enough cases with the depth of invasion only limited to the mucosa, the difference between the expression of 4.1B, 4.1R, 4.1G and 4. 1N with different depth of invasion was not significant (P >0. 05).6. Protein 4.1 expressed in EC9706, EC109, EC1 tumor cell lines.CONCLUSIONS1. The expressed levels of 4.1B, 4.1R, 4.1G and 4.1N in gastric cancer tissues are significantly lower than the control group, suggest that 4.1B, 4.1R, 4.1G and 4.1N are closely related to the occurrence and development of gastric cancer.2. The expressed levels of 4.1B, 4.1G, 4.1N and the differentiation of gastric cancer tissues are significantly correlated. But the expressed levels of 4.1R and the degree of tissues differentiation have no obvious correlation, suggest that the abnormal expression of 4.1B, 4.1G and 4.1N is related to the degree of malignant tumors.3. 4.1B and 4.1N are related to lymph node metastasis of gastric cancer. 4.1G and 4.1R have no obvious correlation with the regional lymph node metastasis, suggest that the abnormal expression of 4.1N and 4.1B is involved with the lymph node metastasis of gastric cancer.4. The expression of 4.1R between gastric cancer and normal tissues is of clear differences, but do not have no obvious correlation with differentiation of the cancer tissues and is not associated with lymph node metastasis, suggest that the abnormal expression of protein 4.1R is an early incident in the occurrence and development of gastric cancer.5. Protein 4.1 express in EC9706, EC109, EC1 tumor cell lines so these cell lines are unsuitable for investigate the function of tumor depression of protein 4.1 by introducting protein 4.1 into them.