Screening For Differentially Expressed Genes Of Gastric Cancer And Relationship Between ERBB2,ERBB3 And Gastric Cancer

Background In the past two decades many important dicoveries and improvements have been made in the gastric cancer research regarding the etiology, pathogenesis and therapeutic methods. Especially with the dicovery of more and more oncogenes and tumor reppressor genes, for example, the identification of CDH1 and human epithelial growth factor receptors family, the carcinogenesis of gastric cancer is becoming more and more clear, and our insight into gastric cancer is deepening step by step. Subsequently, more new innovations and even some breakthrough were made therapeutically,and herceptin- a humanized monoclonal antibody used in the gastric cancer patient with ERBB2 over expression, is the case. Additionally, the development of new approaches to functional genomics, for example the characteristic of"high output"of microarray, has markedly improved our ability to explore molecular alterations underlying gastric carcinogenesis and progression.In the first part of our article, we screened the differentially expressed genes by studing two cases of intestinal type of gastic cancer tissue using cDNA microarray compared with the adjacent normal part. Subsequently immunohistochemistry (IHC) was appllied to investigate the expression of ERBB2 and ERBB3- the up regulated oncogenes which have been identified in the first part. At the same time, real-time quantitative reverse transcriptional ploymerase chain reaction was performed to quantify the relative expression of mRNA of ERBB2 in the gastric cancer tissue. And the genomic mutations of ERBB2 coding sequence of tyrosine kinase were detected by nested PCR and then sequenced. The aim of the study is to elicit the carcinogenesis of the receptor tyrosine kinases (especially ERBB2 and ERBB3) in the gastric cancer, and ascertain whether there exist genomic mutations in the coding sequence of ERBB2 tyrosine kinases. Methods Two pairs of fresh resected samples (intestinal type of gastric caner tissue and the matched adjacent normal tissue according to the Lauren classfication) were appllied to cDNA microarray to screen the differentially expressed genes. The expression of ERBB2 and ERBB3 which were confirmed to be over-expressed in the microarray test, also known as the important members of epithelial growth factor receptor(EGFR) family, were detected in 65 cases of gastric cancer and 25 cases of adjacent normal tissue using immunohistochemistry (IHC). The results of expression of ERBB2 and ERBB3 in gastric cancer tissue were compared with the clinical parameters. Relative quantification of mRNA of ERBB2 in 12 pairs of fresh specimens (the gastric cancer tissue and the matched adjacent noraml tissue) was quantified by real-time reverse transcriptional polymerase chain reaction. The genome was extracted from 55 gastric caner tissues and 25 adjacent normal tissues, and the coding sequence of tyrosine kinase of ERBB2 was amplified by nested-PCR. The PCR products were sent to be sequenced.Results1. The results of gene expression profile by cDNA microarray analysis (1,4000 unique human cDNA sequences): Over 1000 genes were over-expressed in the 2 cases, and 762 genes and 553 genes were under-expressed respectively. Among the two cases, 154 genes were identified as the co-over-expressed genes and 79 genes as the co-under-expressed genes. Oncogenes such as ERBB2 and ERBB3,cell cycle regulating proteins such as cyclinD1 and cyclinB1, molecules of cell metabolism such as CYP3A5,CYP2C8, and cell epithelial cluster antigens such as mucin 1 and mucin 6,were identified.2. IHC demonstrated that ERBB2 and ERBB3 were over-expressed, accounting for 12.3% and 10.8% respectively in the gastric cancer tissues, while there was no positive stain in the adjacent normal tissues. The localization of ERBB2/ERBB3 was mainly the cytomembrane and/or cytoplasma. There were two cases of co-over-expressed ERBB2 and ERBB3, accounting for 3.3%. Furthermore, the rate of positive stain of ERBB2 in the intestinal type of gastric cancer is higher than that in the diffuse type; while the expression of ERBB3 demonstrated the opposite results.3. The results of real-time RT-PCR: The significant difference of the expression of mRNA between cancer tissue and the matched adjacent normal tissue was observed. Of the 12 gastric cancer cases, eight were postive for mRNA of ERBB2;while only three were positive in the 12 adjacent normal tissues. There was 5.76 folds elevation of ERBB2 mRNA in gastric cancer tissue than that in the adjacent tissue by evaluation of Ct. 4. Results of amplification of coding sequence of tyrosine kinase of ERBB2 by nested PCR: Fifty-five cases of cancer tissue got the complete amplification of all the exons of ERBB2 tyrosine kinase for sequencing, and twenty five cases of normal tissue got the complete amplification. No mutations were found in the coding sequence of tyrosine kinase of ERBB2 both in cancer and normal tissues except one missense mutation of TGC-TCC in the intron.Conclusions1. Severe disturbance of genes expression profile in gastric cancer is a common phenomenon. The over-expressed and under-expressed genes extensively and intensively affect cell biocycle, especially the cell growth, proliferation, metabolism, signal communication and so on.2. The different expresson of ERBB2 and ERBB3 detected by IHC in our study suggested that ERBB2 and ERBB3 may play different role in the gastric carcinogenesis of the two gastric cancer types(intestinal and diffuse type according to Lauren's classfication).3. There exists marked activation of ERBB2 in the transcriptive process in gastric cancer tissue than that of matched adjacent normal tissue.4. The analysis of genomic mutation of coding sequence of tyrosine kinase of ERBB2 implied that the genomic mutation of ERBB2 is not common in Chinese gastric cancer patients, or that is at least not a"high molecular incidence"in Chinese gastric cancer patients. Our results seemed to differ from the reports of abroad published papers.