Expression Of Nonsteroidal Anti - Inflammatory Drug - Activated Gene-1 In Human Gastric Cancer Tissue And Its Construction Of Eukaryotic Expression Vector

【Objective】:1.To study the expression of nonsteroidal anti - inflammatory drug - activated gene-1 in human gastric cancer and its relationship with the clinical staging ,differntiation degree ,and lymphatic metastasis of gastric cancer.2.To clone a fragment of NAG-1 gene into an expression vector pEGFP-N1 for expressing NAG-1 protein in vitro.【Methods】:1.The express of NAG-1 mRNA was detected by semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in the cancer tissues and corresponding adjacent normal tissues in 76 gastric cancer patients.2.The expression of NAG-1 protein expression in human gastric carcinoma tissues and normal gastric tissues were detected by S-P immunohistochemistry.3.Using nomal gastic tissue cDNA as template,amplified the NAG-1 gene with RT-PCR.Insert this fragment into the expression vector pEGFP-N1,and identified the positive clone with white and blue assay,endonuclease,PCR and DNA sequenceing identification.【Results】:1.NAG-1 mRNA was positive in both nomal tissues and gastric cancer tissues; expression in gastric cancer tissue significantly was lower than that in nomal tissues(0.5563±0.2181 vs 0.9059±0.0751,P<0.05).NAG-1mRNA expression in well and moderately differentiated cancer cells was significantly higher than those of poorly and non-differerntiated cancer cells(0.6701±0.1637 vs 0.4005±0.1859,p<0.05) , but the expression was not associated with the depth of tumor invasion,lymph node metastasis , and clinical stage (p>0.05).2Immuno-histochemistry showed the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues (p<0.05);and the expression was not relationship with the gender, lymph node metastasis and clinical stage (all p >0.05).3. The expression of NAG-1 mRNA in gastric cancer tissues was lower than that in nomal tissues ;the expression of NAG-1 protein in the cancer tissues was lower than that in the normal tissues.they suggest that the expression of NAG-1 may relationship with the development and progression of gastic cancer.4. The positive clons were identified with PCR,endonuclease and DNA sequencing identification,and they proved that the target fragment of NAG-1 gene had been inserted into expression vector pEGFP-N1 rightly.【Conclusion】:1. The expression of NAG-1mRNA in gastric carcinoma tissues and normal gastric tissues had statistical difference;The expression of NAG-1mRNA had no correlation with the depth of tumor invasion,lymph node metastasis and TNM stage. It is associated with the differentiation of tumor. That suggest the carcinogenesis,progression and invasion of gastric carcinoma had correlation with the NAG-1mRNA expression.2. The expression of NAG-1 protein was significant lower in gastric carcinoma than normal gastric tissues. The expression of NAG-1 protein was associated with differentiation grades.That show NAG-1 protein suppressed the processes of carcinogenesis,progression and invasion of gastric carcinoma. Downregulation of NAG-1 protein promoted the processes of carcinogenesis,progression,invasion and metastasis of gastric carcinoma,Which made the gastric carcinoma cells more malignant and more invasive3. Cloning NAG-1 recombinant plasmid lays the foundation for expressing NAG-1 protein in vitro.