| Objective: High frequency Loss of heterozygosity of gastric carcinoma exists on chromosome 8p21-22. The ESTs (expressed sequence tags, ESTs) associated with gastric carcinoma on this region were screened, which provided basis for further cloning and identify novel genes associated with the development of gastric carcinoma.Methods: EST markers on the chromosome 8p21 region were screened with the BLAST software and 10 EST were filtered out. We selected 4 EST to design primers. 28 cases of gastric cancer surgery samples were collected as cancer groups, corresponding normal gastric mucosa away from the cancer point as the normal control groups. All normal and cancer samples were pathologically diagnosed. The total RNA of samples was extracted out, with reverse transcription polymerase chain reaction (RT-PCR), to screen the differential expression EST. Then in silico clone the EST which expressed differently between gastric carcinoma and normal gastric mucosa through analysis with BLAST soft ware, joint and extend the cDNA of the differential expression EST. Bioinformatics prediction analysis of the cDNA was carried out to identify message of the gene represented by the EST.Results: 10 ESTs in the region chromosome 8p21 screened, 4 of them were selected to design primers. The RT-PCR results showed that EST DA931869 expressed significantly different between gastric carcinoma and normal gastric mucosa by RT-PCR approach(P<0.05), and other three ESTs AI125340 , CR745531 , BG752916expressed no significant difference by statistic analysis. The positive expression rate of EST DA931869 was 89.28%(25/28)in normal gastric mucosa, and 46.43 % (13/28) in gastric carcinoma, reduced or absent expressed compared with in normal, gastric mucosa. The positive rate of ESTDA931869 expressed in well-differentiated gastric carcinoma was 83.33%(5/6), obviously higher than in moderately differentiated gastric carcinoma 57.14%(4/7) and in poor differentiated gastric carcinoma 26.67%(4/15)(.P<0.05).In addition, the positive rate of the EST expressed in lymphoid node metastasis-negative gastric carcinoma was 60.00%(9/15), significantly higher than in lymphoid node metastasis -positive gastric carcinoma 30.77%(4/13)(p<0.05).RT-PCR results showed that the level of the EST expression was 1.192±0.154 in normal gastric mucosa, 0.502±0.171 in gastric cancer. The EST expression in gastric carcinoma is significantly lower than that in normal gastric mucosa (P<0.05). The level of the EST expression in well, moderate and poor differentiated gastric cancer were 0.799±0.167, 0.550±0.112, 0.311±0.208 respectively, which indicated that the levels of the EST expression were significantly related to the degree of the gastric carcinoma differentiation. The level of mRNA expression of EST (DA931869) in lymph node metastasis-positive Gastric carcinoma was 0.266±0.142, and 0.652±0.133 in lymph node metastasis-negative gastric carcinoma group. The discrepancy between the two groups was significant by statistic analysis (P <0.05). Bioinformatics analysis results: This EST located in chromosome 8p21 (24,200-24,215kbp). The EST was included in the contig Hs.637673. We checked it by online digital Northern, which showed that it expressed in human normal tissues. In silico cloning results: Splicing and jointing EST DA931869 on the Internet using BLAST, we obtained a cDNA, 1606bp length, located in the region of chromosome 8p21, homology <50% compared with known genes. PolyA located at 1214-1219 bp.Conclusion:1. The expression positive rate of EST DA931869 was lower in gastric carcinoma tissue than in normal gastric mucosa, reduced or absent expression in gastric carcinoma.2. The cDNA represented by the EST extended to 1606bp through Internet connection, which contains PolyA tail.3. The cDNA abtained has homology <50% with known genes, which indicated that it was a new cDNA sequence of a novel gene locating on chromosomes 8 p21.
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