Study On Relationship Between Survivin And Apoptosis-related Protein Expression And Carcinogenesis And Progression Of Gastric Carcinoma

Inhibitors of apoptosis protein (IAP) have attracted considerable attention as a unique cell death modulating factor. Survivin is a new and the smallest member of LAP family, which was identified by hybridization screening of a human PI genomic library with the cDNA of EPR -1 by Altieri in 1977. Survivin has potential anti — apoptotic activities, and involved in the mitosis and angio-genesis. In contrast to other IAPproteins, survivin was observed to be expressed in fetal tissues and most common human cancers, including carcinomas of the lung, stomach, colon, breast and prostate as well as high - grade non -Hodgkin' s lymphomas, whereas no survivin transcripts were detected in normal , terminally differentiated adult tissues ( except thymus gland and genital gland). It has been known that the overexpressed of survivin in tumor tissues is correlated with poor prognosis of the patients. Survivin can be used as a prognostic factor and a new target in tumor targeting therapy.Tissue microarray/tissue chip, is a newly developed array - bases high throughput technique with a conspicuous characteristic of small volume rich in information that facilitates gene expression investigation of very large numbers of tumors simultaneously. It is suitable to immunohistochemistry, DNA or RNA in situ hybridization with high efficiency and flexibility, and its results are highly uniform with remarkable credibility and comparability. Many reports have demonstrated that tissue microarray/tissue chip is a new powerful technique in pathology of tumors investigation. There have been some reports on investigating the expressions of tumor - related markers in gastric carcinoma and its precan-cerous lesions with the tissue microarray during past five years.Gastric carcinoma is one of the commonest malignancies in China, and e-ven in the world. However, the molecular aspects of carcinogenesis and metastasis of gastric carcinoma remained elusive. Heretofore, there has been few report on investigating survivin, bax and mp53 expression in gastric carcicoma and its precancerous lesions using tissue microarray.ObjectiveThe purpose of present study was to detect expressions of survivin mRNA and protein (a apoptosis -inhibitor) , bax (a apoptosis -promotor ) in gastric carcinoma and their matched adjacent mucosa by using methods of tissue microarray , immunohistochemistry and in - situ hybridization, comparing with the apoptosis - regulator p53 protein expression and clinicopatholgical parameters of the tumor, and to explore the relationship among survivin, bax, mp53 expression and the molecular mechanism of carcinogenesis and progression of gastric carcinoma.Materials and methodsMaterialsTissue source and clinical Materials Ninety - eight cases of surgically removed specimens of primary gastric carcinoma and their metastatic tumors as well as matched adjacent normal mucosa, intestinal metaplasia and dysplasia of the stomach were all retrieved from the pathological files of Cancer Institute, China Medical University and Tumor Hospital of Iiaoning Province. Tumor Stage: 1 case in early stage; 97 in advanced stage including 5 in middle stage. Metastasis: 65 with lymph node metastasis; 4 with liver metastasis (4 of them accompanying with lymph node metastasis) ; 1 with ovary metastasis. Gross type: 6 cases were Bormann II, 84 Bormann III and 7 Bormann IV. WHO' s histological classification: 1 was papillary adenocarcinoma, 9 well differentiated tubular adenocarcinoma, 28 moderately differentiated tubular adenocarcinoma,35 poorly differentiated adenocarcinoma, 4 undifferentiated carcinoma, 4 signet ring cell carcinoma and 17 mucinous adenocarcinoma.MethodsHistopathology All specimens were fixed in 10% formalin ( Containing 0.1% DEPC for in situ hybridization) for 18h to 48h at room temperature and were subsequently embedded in paraffin. Four μm thick sections were cut and then stained by hematoxylin - and - eosin in order to confirm their histological diagnosis. The exact positions of target tissues including primary tumor and metastasis tumor of gastric carcinoma, and normal mucosa, intestinal mateplasis, were marked for preparing the tissue microarray of gastric cancinoma and the precancerous lesions.Construction of tissue microarray A Tissue Array Machine, including Manual tissue puncher/arrayer, Punches/Stylets, Recipient block holders, Depth stop kit, was bought from Steve Leighton Beecher Instruments, USA. Two blocks of tissue microarray were constructed, one containing 124 and another containing 101 small cylindrical samples, 1.0mm each in diameter, from 96 cases of gastric carcinoma and the precancerous lesions. Four μm thick series sections were then cut and stored for use.Immunohistochemistry The Envision immunohistochemical stainning method was used in this study. The rabbit against human survivin and Bax polyclonal antibodies were bought from Maixin Company, Fuzhou. The Envision staining kit and monoclonal antibody against p53 was from Zhonghshan Biotech Company , Beijing. Working dilution for these three antibodies mentioned above was 1: 100. Known positive slides were used for positive control; and for negative control, tissue sections were incubated with 0.01M PBS instead of the primary antibodies.In situ hybridization In situ hybridization for survivin mRNA was performed to all cases. In situ hybridization kit for detecting survivin mRNA was from Boster Biotech Company, Wuhan.Criteria for judging results Brown grains existing in cytoplasm or nucleus were considered as positive for survivin protein and mRNA expression, in cytoplasm for bax and in nucleus for mp53 protein expression. Slides were scoredsemi - quantitatively based on staining intensity and distribution. Positive rate was assessed by the percent of positive cells in all counted cells (in 2 random chosen fields, 400 x ) and grades are; negative ( - ) , positive rate < 5% ; weakly positive ( + ) : 5 - 25% ; moderately positive ( ++ ) : 25 - 50% ; strongly positive ( +++ ) : > 50%.Statistic analysisAll data were disposed by SPSS 12.0 statistical software to analysis the statistical differences between different groups via chi - square test and correlation coefficient. A value of P <0. 05 was considered significant, and P <0. 01 remarkably significant.Results1. The positive rate of survivn gene encoding protein in gastric cancer tissues (80. 6% , 89/98) was significantly higher than those in adjacent normal mucosa(3. 9% ) , intestinal metaplasia (91. 4% ) and dysplasia( 100% ), p < 0.01. The positive rates of survivn protein expression in tumors with metastases ( in lymph node metastasis 86.2% , linver metastasis 100% and ovarian metastasis 100% ) was statistically higher than in tumors without metastasis (64.3%), p <0. 05. Survivin protein expressionin gastric cancer tissues was closely correlated with the mp53 protein expression, p <0.05o survivin protein expression was not related to clincopathological stage, histological classification, lauren"s type and gross type of gastric cancer, p >0.05.2. The positive rate of survivn mRNA expression in gastric cancer tissues (81. 6% ) was significantly higher than in normal mucosa(6. 5% ) , intestinal metaplasia(92. 6% ) and dysplasia( 100% ) , p < 0. 01. The positive rates of survivn mRNA expression in tumors with metastases ( in lymph node metastasis 87. 5% , linver metastasis 100% and ovarian metastasis 100% ) was statistically higher than in tumors without metastasis(62.5 % ) , p <0.05. Survivin mRNA expression was closely related to the protein expression in gastric cancer tissues, p<0.01, R=0.550. However, survivin mRNA expression was not related to clincopathological stage, histological classification, lauren" s type andgross type of gastric cancer, p >0.05.3. The positive rate of apoptosis - inducing protein Bax expression was 17.1% (17/96) , statistically lower than that in normal adjacent normal mucosa (51% ) , intestinal metaplasia(69.2% ) and dysplasia(75% ), p <0.01. Bax expression in poorly differentiated adenocarcinoma(40% ) was significantly lower than in well differentiated adenocarcinoma(58.4% ) , p <0.01. Bax expression was not correlated to clincopathological stage, histological classification, gross type and metastasis of gastriuc cancer, p >0.05. There was no significant correlation between bax expression and survivin and mp53 expression, p > 0.05.Conclusions1. In pathology of tumor investigation, tissue microarray is a new powerful technique with a conspicuous characteristic of small volume rich in information that facilitates gene expression investigation of very large numbers of tumors simultaneously , and must be widely employed in tumor pathologic diagnosis and research in near future.2. Survivin gene was a lock -up gene in normal human tissues. It was activated and take a part in tumorigenesis and progression of gastrc cancers. mR-NA and protein of survivin can be a ideal marker for early diagnosis of gastrc carcinoma and the metastasis. It was surely helpful in elucidating the mechanism of tumorigenesis, and providing a new tumor marker and a biological tretment target.3. p53 and survivin genes might share the same mechanism in regulating cell poptosis. P53 gene was mutated frequently in most malignant tumors, meanwhile, abnormal expression of survivin gene was also detected in most cancers. It was concluded that mutation of p53 gene may lower its down - regulation to survivin expression.4. Apoptotsis inducing protein Bax was important to maintain the normal structure and function of gastric mucosa. The abnormal expression of Bax was correlated with the tumorigenesis and progression of gastric carcinoma. Bax pro-...