Study Of Molecular Mechanisms In Gastric Carcinoma And The Relationship With Apoptosis Using CDNa Microarray And Tissue Microarray

Gastric carcinoma is one of the most common malignant tumors in our country. The occurrence and development of gastric carcinoma was a multi-step and extremely complex process in which many factors participated and involved a cascade of genetic alterations. However, the molecular mechanism of carcinogenesis is not clear. In this study, to explore application value of cDNA microarray and tissue microarray technology in high-throughput screening gastric carcinoma-related genes and molecular pathophysiology, the differentially expressed genes in gastric carcinomas and the adjacent noncancerous gastric tissue were investigated. To provide theoretical and experimental basis for further investigating, diagnosing and treating gastric carcinoma, apoptosis-related genes and key effectors of apoptosis in the process of gastric carcinogenesis were also observed in gastric carcinoma and precancerous lesions.In this present study, a gene expression profile chip consisting of 4096 human genes was prepared using a spotting robotics onto a kind of chemical-material-coated-glass slide. Total RNA was extracted from gastric adenocarcinoma and noncancerous gastric tissues using one-step method. The fluorescent probes were prepared from cDNA labeled with cy3-dUTP and cy5-dUTP using reverse transcription method for these two kinds of tissues respectively and hybridized with the cDNA microarray, followed by scanning microarray for obtaining fluorescent image signal. The differentially expressed genes were determined using ImaGene 3.0 software. Cathepsin D and CD9 in 21 cases of gastric carcinoma, 19 cases of adjacent noncancerous gastric tissue and 21 cases of normal gastric mucosa samples were detected using immunochemistry on tissue microarray and the results of gene microarray were partially verified; 46 cases with gastric cancer, 20 cases withprecancerous lesions of gastric carcinomas and 24 cases with normal gastric mucosa were adopted. TUNEL (TdT-mediated dUTP nick end labeling) method and immunohistochemical techniques were used to detect the state of apoptosis and proliferation of gastric carcinoma. The expression of apoptotic associated genes p , bcl-2, and c- myc was also analyzed by immunochemistry; The key effectors of apoptosis, such as Caspase —3 and Caspase — 8 in gastric carcinoma, adjacent noncancerous gastric tissue and normal gastric mucosa samples were detected by S-P immunohistochemistry method on tissue microarray.The results are as below:(1) Results of cDNA microarry screen: In 4096 genes, the differentially expressed genes between gastric adenocarcinoma and normal gastric mucosa were 190 Cy5 / Cy3 >2 or <0.5(4.6%). Among them, there were 85 up-regulated genes (2.1%), 105 down-regulated genes (2.6%), 100 known genes (Gene Bank Registered) and 90 unknown genes. These differentially expressed genes compared with normal tissue were mainly involved in metabolism, signal transduction pathway, immunity, proliferation, cell cycle and apoptosis. The genes were closely related to cellular physiological functions, such as ion channel, transport protein, metabolism and others showed a trend toward down-regulating; There were both up-regulated and down-regulated genes in which was related to mucosa development and differentiation related genes such as regulating cell cycle proteins expression genes, apoptosis related genes, development related genes, pro-oncogenes and tumor suppressor genes, the genes relative to DNA binding, transcription and protein translation and synthesis, and the number of up-regulated was similar to down-regulated genes.On the other hand, the genes related to cellular skeleton, migration, signal tansduction proteins had some up-regulated ratio.There were 49 differentially expressed genes Cy5 /Cy3>4 or<0.25 between gastric carcinoma and normal tissue. Among them, there were 23 up-regulated genes which was related to cellular signal transduction proteins (AK001903, NM004369,NM₀05796 and NM₀00582) (GeneBank ID), cellular skeleton and migration(NM₀00089, NM₀03118, NM₀00393, NM₀00090) and related metabolism(NM₀12145 and NM₀01428) ; there were 26 down-regulated genes which wasmainly related to immunity (NM₀01185, NM₀01823, NM₀00140, NM₀04867and NM₀25190 ) , signal tranduction proteins ( NM₀03558 andNM₀02731) ,pro-oncogenes and tumor suppressor gene (NM001910, BCO 11762 andD42047) and development (NM₀05142, NM₀02443 and NM₀04081) .In thisstudy, apoptosis related genes were down-regulated (NM₀14757^ NM₀13995) ,butapoptosis-inhibiting genes were up-regulated (AK024172> AF311912) .(2) The results of immunochemistry on tissue microarray: Cathepsin D expression was lower in gastric adenocarcinoma compared with normal tissue (P<0.01) (cDNA microarray hybridization ratio 0.117), but CD9 expression in gastric adenocarcinoma was significantly higher than that in normal tissue(cDNA microarray hybridization ratio 2.803). The trend of differentially expression was consistent with the results of cDNA microarray.(3) The apoptotic cells in normal gastric mucosa usually located in superficial layer and proliferative cells located in basal part. During the gastric carcinogenic process, the apoptotic and proliferative indexes increased simultaneously in early stages. When precancerous lesions occurred, proliferative index (PI) was further increased but apoptotic indexes (AI) was decreased. AI in precancerous lesions was significantly higher than that in gastric carcinoma and normal mucosa (p<0.001), while AI in gastric carcinoma was markedly lower than that in normal mucosa (p<0.001); PI in gastric carcinoma was significantly higher than that in precancerous lesions and normal mucosa (p<0.001), while PI in precancerous lesions was obviously higher than that in normal mucosa (pO.OOl). The ratio of AI and PI was lower in gastric carcinoma than that in precancerous lesions and normal mucosa, respectively (p<0.001).(4) The positive expression rates of p53, bcl-2 and c-myc protein were 60.9%, 67.4% and 73.2%, respectively, in gastric carcinoma, they were all higher than those in normal mucosa (p<0.01). The positive expression rates of p53, bcl-2 and c-myc protein were 40.9%, 95% and 58.8%, respectively, in precancerous lesions (p<0.01), all higherthan those in normal mucosa (p<0.01).The positive rate that three kinds of proteins expressed simultaneously was 36.96%-? 30.0% in gastric carcinoma and precancerous lesions respectively, There was significant difference compared with the normal tissue (PO.01); AI in the group with positive expression of p53 and bcl-2 protein was lower than that in the group with negative expression (p<0.05); AI in the group with positive expression of c-myc protein was higher than that in the group with negative expression(5) The results of S-P immunohistochemistry method on tissue microarray: The positive rates of Caspase3 and Caspase8 in gastric carcinoma were 28.6% and 27.8% respectively, and both was lower than those in normal tissue (PO.01); the positive rates of these two proteins in precancerous lesions were 52.6 % and 50.5% respectively,and both was higher than these in normal tissue (P<0.01).Our conclusions:1 -. The occurrence of gastric carcinoma was involved the changes at polygene expression level, which was mainly involved in metabolism, signal transduction pathway, immunity, proliferation, cell cycle and apoptosis. cDNA microarray technology can provide special and reliable data for gastric carcinoma related gene analysis, and effectively screen novel gastric carcinoma associated genes.2 ^ During the canceration of gastric mucosa, not only proliferation is active, but also apoptosis is under inhibiting condition. The dysequilibrium of apoptosis and proliferation play an important role in occurrence and development of gastric carcinoma.3 ^ P53 and Bcl-2 proteins have inhibitory effects on apoptosis, but c-myc protein promotes apoptosis , there is a close relationship between abnormal apoptosis and occurrence of gastric carcinoma .4, The down-regulating of caspase-3 and caspase-8 protein is associated with abnormal apoptosis. The abnormal decrease of caspase-induced apoptosis may be involved in occurrence of gastric carcinoma.5> cDNA microarray, as rapid, high-throughput and sensitive technology, is a stable and high effective method in screening gastric carcinoma associated genes.6> Tissue microarray has many advantages such as high-throughput, large samples, time-saving, speed, easy designing experimental control and reliability. It is development and extension of cDNA microarray.