The studies on regulating the expression of PTEN and Livin genes in gastric carcinoma cell line BGC823 and characterization of their bioactivity

Abstract

Objective To study the effect of simultaneously increasing PTEN gene expression and inhibiting Livin expression on the proliferation, apoptosis, invasion and metastasis of gastric carcinoma cell line (BGC823) in vitro. Methods To construct the eukaryotic expression vector pCL-neo-PTEN and siRNA vector pRNAT-U6, 1-Livin. The siRNA expression unit against Livin gene (siLivin) was cut from pRNAT-U6, 1-Livin vctor, and then inserted into pCL-neo-PTE to construct the recombinant vector pCL-neo-PTEN-siLivin. Then, pCL-neo-PTEN-siLivinp, pCL-neo-PTEN, pRNAT-U6, 1-Livin, pCL-neo,pRNAT-U6. 1 were respectively transfected into the gastric carcinoma cell hne (BGC823) with LipofectAMINE 2000TM. The mRNA and protein expression level of PTEN and Livin genes in each group cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, Cell proliferation curve, the activity of Caspase-3 and 9, cell apoptosis, ability of invasion and metastasis were detected in vitro. Results Recombants vector of pCL-neo-PTEN, pRNAT-U6, l-Livin and pCL-neo-PTEN-siLivin were constructed successfully. After transfected with pCL-neo-PTEN-siLivin, the mRNA (0. 897±0. 112) and protein expression level of PTEN in BGC823 cells were rised,meanwhile, Livin genes were silenced (0. 071 ±0. 009). And characterization of the regulated cells bioactivity has improved, such as the reduced proliferation ,higher apoptosis ( 16. 72±1.84) ,more effective activity of Caspase-3 and 9 ( 1.88 ±O. 21,1.07 ± 0. 18 ), fewer cells penetrating the Matrigel (23. 1 ± 3. 5 ). There were significant differences between transfected groups and control groups ( P < 0. 05 ). And the inhibiting effect on the BGC823 cells' proliferation and metastasis by increasing PTEN expression and silencing Livin simultaneously was better than that only by regulating PTEN genes or Livin genes alternatively. Conclusion The PTEN gene upregulated and the Livin gene silenced at same time could contribute to reduce the proliferation and metastasis in gastric carcinoma cell line BGC823. Key words: PTEN; Livin; Gastric carcinoma cell line BGC823; Regulating; Growth inhibition