Inhibitory Effects of Cotransfection of Two Short Hairpin RNA on Expression of BAG-1 Subtypes in Gastric Carcinoma

Objective To construct the eukaryotic expression plasmids of short hairpin RNA(shRNA) specific for Bcl-2 associated athanogene 1(BAG-1);and to observe their inhibitory effects on the main subtypes expression of BAG-1 gene in human gastric carcinoma SGC-7901 cells. Methods Two distinct interference segment A and B of 64nt oligonucleotides which were located on two common exons of BAG-1 gene P29, P33, P46 subtypes and one negative control segment S, were designed and directionally cloned into eukaryotic expression plasmids pGenesil-1 containing kanamycin resistance gene and GFP to target the human BAG-1 mRNA coding region. Lipofectamin TM 2000 was used to cotransfect plasmids into SGC-7901 cells. Negative plasmid-transfected, untransfected SGC-7901 cells and transfected interference plasmids pGenesil-BAG-A and pGenesil-BAG-B served as negative, blank, and interference control respectively. Interference effects were measured by RT-PCR and Western blot. Cell proliferation was detected by MTT assay and cell growth curve was drawn to analyze the inhibitory effects. Results The clone was verified through restriction enzyme digestion and sequence analysis of hairpin-siRNA expression DNA segment and adjacent vector sequence. The transfection rate in SGC-7901 cells was 40%-50%. Compared with the untransfected control, the negative control cell BAG-1's gene expression showed no obvious difference. However, the mRNA and protein of BAG-1 in SGC-7901 cells was significantly inhibited by BAG-1 shRNA (both P<0.01). The inhibition rate of mRNA was (63 ±9.8)%. The inhibition rate of P46 and P32 BAG-1 proteins were (94.3±1.08)% and (66.1±6.5)%, respectively. The proliferation of cells was inhibited significantly compared with the control group. Conclusion The vectors containing hairpin-siRNA expression DNA segment are successfully constructed, and cotransfection plasmids of pGensil-BAG1-A and pGensil-BAG1-B can significantly inhibit BAG-1gene's main subtype expression and cell proliferation of human gastric carcinoma SGC-7901 cells.