| ABSTRACTGastric carcinoma is one of common malignancies and seriously threaten thehealth of mankind. The abnormality of many oncogenes and suppressor gene areclosely related to gastric carcinogenesis and development. However at present it isseldom reported with regard to some important target genes,which are of significancein the prevention and treatment of gastric carcinoma. In order to do this, it is necessaryto search for gastric carcinoma associated genes. Our previous work have found somedifferentially expressed genes with Suppression Subtractive Hybridization . Then ,wedetected and confirmed the differentially expressed genes in dysplasia, early gastriccancer ,advanced gastric cancer ,metastatic carcinoma and corresponding normalgastric mucosa by using cDNA array, dot blot hybridization technology etc. Ribosomalprotein S12(RPS12) gene is one of the most important differentially expressed genesamong them. It was overexpressed in the samples of the different stages . It is animportant differentially expressed gene in different stages of gastric carcinogenesis anddevelopment. It is necessary to confirm its contribution to gastric carcinogenesis anddevelopment. Based on our previous work, in order to further our study about the roleof RPS12 in gastric cancer,we intend to construct RPS12 specific shRNA expressionvector and transfect it into gastric cancer cell,then confirm the silencing of RPS12gene. We observe biological characteristics of gastric carcinoma,such as apoptosis, proliferation, and explore its molecular mechanism. Our research would deepen thecomprehension of molecular mechanism about gastric carcinoma and we expect todiscover a biomarker for the diagnosis and prognosis of gastric cancer.Materials and methods1．Materials: Gastric cancer cell line BGC823,RPMI1640 medium,TRIZOLReagent,Taq DNA polymerase,Reverse transcription kit,plasmid extractionkit(Qiagen company); liposomal transfection kit(invitrogen company); BamH I,Hind (?) EcoR (?) (Takara company); PI,JM109 competent cell(Promega) etc.2．Methods: We constructed RPS12 specific shRNA expression vector by usingrecombinant DNA technology, transfected it into gastric carcinoma cell with genetransfection technology,with the scramble-shRNA as control . RPS12 gene expressionof BGC823 cell was determined by RT-PCR to confirm the effect of interference.Inorder to analyze the effect of RPS12 gene on the biological characteristics of gastriccancer cell, we detected the proliferation and apoptosis of gastric cancer cell after RNAinterfere by MTT and FCM .At the same time, we evaluated the expression ofapoptosis associated genes to explore the possible molecular mechanism, by whichRPS12 gene influence the apoptosis of gastric cancer cell.Results1．The effect of RPS12 RNAi on RPS12 mRNA expression in gastriccancer cells cells:The results of RT-PCR showed the RPS12 mRNA expression was obviouslydown-regulated on day 3,5and7 after transfection of RPS12-shRNA expressionvector compared to control group , especially on day 5．2．The effect of RPS12 RNAi on the proliferation of gastric cancercells:The results of MTT indicated that the proliferation ability of gastric cancer cell decreased on day 3,5,7and9after transfection of RPS12 shRNA ,compared tocontrol group, especially on day 5．3．The effects of RPS12 RNAi on apoptosis of gastric cancer cells:RPS12 RNAi induced the apoptosis of gastric cancer cell. After RNAi, the rate ofapoptosis in the cells reached 17．84%.4．The effect of RPS12 RNAi on Bcl-2, Bax mRNA expression ingastric cancer cells:The expression of Bcl-2 mRNA was obviously down-regulated and no change wasfound for Bax mRNA expression on day 5 and 7 after transfection of RPS12 shRNAcompared to control group.ConclusionBy silencing RPS12 gene with RNAi, our research demonstrated that RPS12 genemay promote proliferation and inhibit apoptosis of gastric cancer cell .In the meantime,We found that RPS12 gene may act as an up stream gene to regulate Bcl-2 geneexpression and influence the apoptosis of gastric cancer. |
PDF Full Text Request