| Background:Gastnc cancer is a kind of high malignant tumor At present, its incidence and mortality rate is still high in the world Especially in our country, gastric cancer is still a major killer Gastric carcinogenesis results from the accumulation of a series of genetic and biochemical changes in normal mucosal cells, and it is important that the protooncogenes are activated and tumor-suppressor genes lose functions in this process Despite the increase in our knowledge of oncogenes and tumor suppressor genes associated with gastric cancer over the past decades, the specificity of these genes are poor for gastric cancer, the molecular events leadings to the formation of gastric cancer are still not well understood Therefore, identication of genes that are involved in the transition of non-tumorigemc cells to malignant cells can be expected to help us understand the molecular mechanism underlying tumor formation and provide insight into gene diagnosis and treatment of gastric cancer and precancerous conditionsObjectives:To search for new human gastric cancer-associated susceptible gene for early diagnosis and treatment of gastric cancerMethods:1 Construction of cDNA Library Total RNA and mRNA was extracted and converted to blunt-ended, double-stranded cDNA by Oligo(dT)-mediated reverse transciption followed by adapter addition Column chromotography isolates residual adapters and small bands Ligation of cDNA to the vector7and in vitro packaging of ligated cDNA and introduction into E ccli Y1090 Titer and amplify the phage2Cloning gastric cancer related candidated homologenes Previously, W123, a EST fragment derived from normal gastric and paracarcinoma tissue, was cloned using differentiated-display PCR technique A few homologous EST sequences were captured when processing similarity search in human EST database To search for additional W123 homologous genes in gastric tissue, we designed a primer for 3'-RACE from highly conserved region among the above ESTs Using this 3'primer and another 5' primer provided by 3'-RACE kit, we do rapid amplification of eDNA 3'end The cDNA bands got from 3'RACE were cloned into pGEM T-Easy vector, and the positive clones were confirmed by EcoR I enzyme digestion and T7/SP6 PCR amphcation Then ,the sequences of inserted fragments were analyzed The bioinformatics source was utilized to get more information of these cDNAs, and submitted them to Genbank3Testifying cDNAs expression in different gastric and other tissues Northern blot and C-PRJNS technology are applied to detect the expression of these bands in gastric cancer and normal tissues, combination of virtual Northern and multiple tissues Northern blot, expression of cDNAs in multiple normal and carcinoma tissues were analyzed Image analysis sofeware was used to quantitate the expression of cDNA bands, and mean OD was used as the quantitative indexResults:1The cDNA library has 3 2 X 1 O6recombinants,the recombining ratio is96% The titer of amplified library is 12 Xl 012p~ml Identification of theinsert fragments of the recombinant phage DNA are between 0 5---3 5kb,average size is 1 3kb2Seven cDNA fragments with polyA tail were cloned, polyA contains 20-30 "A" , sometimes AATAAA is found among cDNAs Comparison analysis with Genbank demonstrated all these ESTs may represent 7 unknown genes8contaimng a fragment of common sequence They were named W13 ~ W25.~ W3 1 ... W4 1~ W5 1. W63 and W74, respectively These bands were submitted to Genbank dbEST database and already admitted, and Accession Numberwere AF332603~ AF332604~. AF332605N AF325202~. AF332606.. AY0326l5~, AY0326163 Northern blot revealed Wi 3 W3 1 .~. W4 1-. W5 land W74 presented higher expression in gastric cancer tissue than normal tissue, but W63 expressed reversely W3 1, W4 1 and W63 have three transcnpts, the estimate mRNA full length is 1 2-~6 0kb, W51 is 3 5kb, the length of W74 is between 3 0-5 0kb Multiple tissue Northern blot revealed W4 1 presented a high...
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