| ObjectGastric carcinoma is one of the major malignancies considerably threatening human health in China, widespread invasion and metastases to vital organs are the leading causes of mortality. Early diagnosis and prevention of gastric carcinoma from metastasis have become the greatest challenge to improve 5-year survival of patients with gastric carcinoma. In recent years, it has been recognized that oncogenesis and metastasis is a progressive course involving multiple genes interacting with each other. A dynamic and integrative dissecting of genetic changes in tumor progression thus is essential for tumor therapy/prevention. Gene microarray provides an ideal technical support for profiling gene expression involved in oncogenesis /metastasis of gastric carcinoma, by comparing genes differentially expressed among individuals, tissues, and under different stimulating conditions, identify genes groups which are specifically expressed in different individuals/tissues/stimulations, microarray analysis is also able to define the dynamic characteristics and patterns of genes in different groups.In this study, microarrays containing 14784 individual full length cDNAs from Homo sapiens were applied to screen genes differentially expressed in clinical samples of resected gastric carcinoma and their adjacent normal gastric tissue, as well as samples of primary /metastatic gastric carcinoma. Furthermore, we have confirmed differentially expressed genes in gastric carcinoma and nodular metastasis tissue by RT-PCR, immunehistochemistry and western blot. Our study have identified several genes involved in the oncogenesis/metastasis of gastric carcinoma, which may potentially provide therapeutic targets for the prevention of metastasis and improve long-term survival of patients with gastric carcinoma.Methods1 Collection of samples and clinical/pathological data:6 patients of gastric carcinoma entered into this study were from the First Affiliated Hospital of Dalian Medical University, tumors were surgically resected and pathologically verified, 4 cases were diagnosed as nodular metastases, 1 case as hepatic metastasis. Primary gastric carcinoma, corresponding noncancerous gastric mucosae and metastatic carcinoma were obtained instantly after resection, kept in liquid nitrogen for later usage. None of patients has recieved pre-surgical chemotherapy or radiotherapy.2 Preparation of cDNA Microarrays.14K cDNA microarray filter (provided by Research Genetics, Inc., Shanghai) was adopted. In order to monitor the liability of hybridization data of microarray, housekeeping genes were served as positive control, bacteria genes (6) as the negative control, sample solution were used as blank. Human 14KcDNA microarray contains 14784 genes in total, with the matrix of 18*18*48 points (sub-matrix), point interval of 230μm.3 RNA ExtractionTotal RNA were extracted from primary gastric carcinoma, corresponding noncancerous gastric mucosae and metastatic carcinoma, respectively, with Trizol one-step method, RNAs were further purified with QIANGEN Rneasy Kit. Agarose gel electrophoresis was used to analyze the intensity ratio between 28s and 18s to evaluate the quality of total RNA.4 Labeling and Hybridization cDNA probes were reversely transcribed from mRNA according to the methods by Schena et al. Cy5-dUTP and Cy3-dUTP were used to label mRNA probes from different tissues. After the degeneration of hybridization soluteion of mixed probes and microarray, hybridization soluteon was dropped onto the sampling area of microarray, covered with a cover slip, placed into the hybridization cabin, sealed with parafilm, and then hybridized in 42℃water bath for 16-hour.5 Analysis of resultsThe microarrays were scanned with Confocal fluorescence scanner and the data was analyzed by Pix QuantArray software to plot the value of Cy3 and Cy5, the ratio of Cy3/Cy5 was calculated. Result analysis: (1) RNA extraction were satisfactory; (2) Intensity of either Cy3 or Cy5 must be more than 800; (3) Genes were regarded as differentially expressed if the ratio of absolute value of natural logarithm of Cy3 and Cy5 (log Cy3/logCy5) is more than 2 or less than 0.5.6 RT-PCR verification of part of differentially expressed genes.RNAs of gastric tumor and nodular metastatic tissue were extractd to undergo reverse transcription from primer under normal conditions, and then amplified with PCR. The expression of differentially expressed genes was detected in gastric carcinoma and nodular metastatic tissue, results of gene chips were verified.7 Verification at protein level of EphB4 (one of differentially expressed genes).Ephb4, which was identified by microbaray as differentially expressed gene, was further verified by immunohistochemistry and western blot analysis. The correrelation between EphB4 expression in normal gastric mucosae, gastric carcinoma at different stages and nodular metastatic tissues, with the clinical/pathological factors of gastric carcinoma were analyzed.8 Statistical analysisSPSS 10.0 software was adopted. Data were analyzed by using X2 test, with Fourfold table method being used to calculate the exact probability. P<0.05 was regarded as statistically significant.Results1 RNA quality controlTotal RNAs extracted from gastric carcinoma tissue and corresponding noncancerous gastric mucosae were between 50-100ug. Agarose gel electrophoresis showed clear bands of 28s and 18s rRNA, suggesting satisfactory purification and integrality of RNA.2 Analysis of differentially expressed genesDifferentially expressed genes in carcinoma and normal mucosae samples were screened against gene chips containing 14784 genes to identify genes responsible for oncogenesis and metastasis in gastric carcinoma. Compared with normal gastric mucosae, 40 differentially expressed genes were detected in 6 cases of gastric carcinoma tissue, including 21 genes with up-regulated expression and 19 genes with down-regulated expression. Compared with primary gastric carcinoma, 46 differentially expressed genes were detected from 4 cases of nodular metastatic tissues, including 18 genes with up-regulated expression and 28 genes with down-regulated expression. 178 differentially expressed genes were detected from hepatic metastatic tissue, including 114 genes with up-regulated expression and 64 genes with down-regulated expression.3 Verification of part of differentially expressed genes by RT-PCRIn order to verify the differentially expressed genes detected from cDNA chip experiment, 7 genes with most altered expression were chosen from 6 cases of gastric carcinoma to be verified by RT-PCR, of which 5 genes were confirmed to be up-regulated (S100A6, S100A11, ETV4, CDH17 and Ephb4) and 2 genes to be. down-regulated (NK4 and PPP2R1B). Furthermore, 2 up-regulated genes (S100A4 and Ephb4, respectively) in nodular metastasis were verified by RT-PCR to be in accordance with the pattern of gene microarray detection.4 Verification at protein level of gene EphB4 (one of differentially expressed genes).Imunohistochemistry staining localized the expression of EphB4 to the plasma of tumor cells and endothelial cells of blood vessels. EphB4 were stained positive in 23 out of 40 cases (57.5%) of gastric carcinoma, while only positive in 6 out 40 (15%) of the corresponding normal gastric mucosae, with significant difference (p<0.05). Imunohistochemistry staining showed EphB4 expression levels were positively correlated to the extent of invasion, Lauren classification and nodular metastasis of gastric carcinoma, while were not correlated with sex, age of patients or the differentiation degree of the carcinoma cells.Conclusions1. Profound alterations of gene expression were observed between gastric carcinoma and normal gastric mucosae, as well as nodular/hepatic metastases and primary gastric carcinoma. Microarray analysis is efficient in the screening of differentially expressed genes related to gastric carcinoma and its nodular/hepatic metastases. Our gene chip data implicate oncogenesis, progression, and metastasis of gastric carcinoma is a progressive course and the consequence of accumulation of multiple genetic changes.2. As proved by RT-PCR on partial genes that are differentially expressed, the altered expression of S100A6, S100A11, ETV4, CDH17, NK4, PPP2R1B, S100A4 and Ephb4 might be involved in the oncogenesis and progression of gastric carcinoma.3. EphB4 is highly expressed in gastric carcinomas, with significant correlation to the extent of invasion, nodular metastasis and Lauren classification. EphB4 may be adopted as a new index to assess the malignant degree, and a potential therapeutic target for gastric carcinoma.
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