| IntroductionGastric cancer is the first leading cause of cancer death in China. Epithelial dysplasia of gastric mucosa is one of well known precancerous lesions and is considered as one of the earliest phenotypic changes in the cascade of events from normal mucosa to gastric cancer. The molecular mechanism for gastric car-cinogenesis has not been elucidated. Losses or rearrangements of genetic material, promoter methylation silencing, and expression level alteration occur frequently during gastric carcinogenesis. These events can be revealed by identification of differentially expressed genes using suppressive subtractive hybridization ( SSH). However, in tissue, dysplasia are dispersedly distributed in lesion tissue and contain low relative abundance of dysplasia cells compared with corresponding normal cells, therefore, microdissection of tissue sections and cytologi-cal preparation must be used for isolation of homogeneous cells. Here, in combination with whole transcriptome amplification (linker adaptor - PCR, cDNA -PCR) as described by cDNA -RDA (representational difference analysis) , we established a new microdissection - cDNA PCR - SSH method to construct subtracted cDNA libraries between microdissected preneoplastic lesion and normal tissue of gastric epithelium. Furthermore, part of clones from subtractive libraries were sequenced and made homologous analysis. Then, we screened associated genes of gastric dysplasia and further investigated their expression in gastric carcinoma with different stages using dot hybridization and microarray. These will provide important clues for identifying associated oncogenes or tumor suppressor genes of gastric carcinogenesis. ACE2 and SDFR1 gene were studied u-sing RT - PCR and mRNA in situ hybridization to speculate their roles in gastric carcinogenesis and development.Materials and Methods1. Microdissection - cDNA PCR - SSHRelatively pure dysplasia and normal tissue were procured by manual mi-crodissection. Whole transcriptome amplification (linker adaptor - PCR, cDNA- PCR) as used by cDNA - RDA was employed to amplify total mRNA from little genetic material. Firstly, cDNA was digested with Mbo I and ligated with adaptor. Then, whole cDNA was amplified by the way of linker adaptor - PCR with special adaptor as primer. Amplified products were used to carry out forward (dysplasia as tester, normal tissue as driver) and reverse (normal tissue as tester, dysplasia as driver) SSH. Subtracted cDNA fragments were cloned into vector, screened, sequenced, and made homologous analysis. If clone had new cDNA sequence, it was submitted to GenBank.2. Dot hybridization26 positive clones were dotted on hybridization membrane using 96 well dot- blot system. Their expression was detected using dot hybridization in gastric carcinoma with different stages, including 1 dysplasia, 1 early carcinoma and 4 advanced carcinoma. Differentially expressed genes were screened compared with corresponding normal tissue.3. MicroarraySelf-made gastric carcinoma-associated gene microarray was used to study dynamically and integrally gene expression profile in the process of gastric carci-nogenesis and development, including 26 screened positive clones. Neighbouring stages were designed as experimental and control group, respectively, and their differentially expressed genes were screened.4. RT-PCRThe expression of ACE2 and SDFR1 gene was detected with (3 - actin as internal control in 30 advanced gastric carcinoma and their corresponding normal tissue, gastric carcinomous cell MKN1 and BGC823, hepatic carcinomous cell BEL -7402. Primers were designed according to Primer 3. 0 software.5. mRNA in situ hybridizationThe expression of ACE2 and SDFR1 gene was detected with their clone fragments as probes in paraffin sections of gastric carcinoma with different stages, including 3 normal mucosa, 3 dysplasia, 3 early carcinoma, 3 advanced carcinoma, gastric carcinomous cell MKN1 and BGC823, hepatic carcinomous cell BEL-7402.Results(l)Part1...
|
PDF Full Text Request