P-0030 CypA Plays Roles in the Proliferation of Gastric Carcinoma Cells

Abstract

ABSTRACT Introduction Cyclophilin A (CypA) is a member of the peptidyl-prolyl isomerases, the primary intracellular receptor of the immunosuppressant cyclosporin A (CsA). CypA plays an important role in life activities and participate in a variety of disease processes. Recent evidence points to possible roles for CypA overexpression in tumorigenesis. However gastric carcinoma was rarely involved in CypA research so far. Methods In this research, using immunohistochemistry assay we confirmed the overexpression of CypA in gastric carcinoma tissues at protein level. The mRNA level of CypA in gastric carcinoma cell lines are also detected by Real-Time PCR. Results Compared with normal gastric epithelial cell line GES-1, the mRNA levels of CypA in gastric carcinoma cell lines SGC-7901, MKN-45, BGC-823 and MGC-803 were 1.29, 1.46, 1.54 and 1.55 fold high respectively (Fig.1). To investigate the roles CypA plays in the occurrence and development of gastric carcinoma, we constructed 4 RNA interference vectors: pRNA-CypA shRNA 1-4 and confirmed the positive clone by PCR with pRNA primer, then sequenced the plasmid DNA for additional verification. BGC-823 cells were grown to 70% confluency in 24-well plates and transfected with 0.5ug pRNA-CypA shRNAs using Lipofectamine LTX (Invitrogen). At 48 and 96 hours following transfection the mRNA level of CypA in these cells were detected by Real-Time PCR. The results showed us that after transfection with pRNA-CypA shRNA3, BGC-823 cells yielded ∼80% and ∼30% CypA knockdown at 48h and 96h respectively compared with untreated BGC-823 cells; with pRNA-CypA shRNA2 yielded ∼27% and ∼35% CypA knockdown at 48h and 96h respectively; pRNA-CypA shRNA1 and pRNA-CypA shRNA4 had little RNA interference effects. A transient increase of CypA mRNA level occurred in transfection, and reduced to the normal level at 96h after transfection. We collected the BGC-823 cells at 48h with the best RNAi efficiency by pRNA-CypA shRNA3 plasmid to perform flow cytometric analysis of cell cycle. From the cell cycle distribution, cell populations of S & G2/M phase (M2 & M3) in untreated BGC-823cells, BGC-823cell transfected with pRNA plasmid and CypA knockdown (transfected with pRNA-CypA-shRNA3 plasmid) BGC-823cell were 45.3%, 49.7% and 33.0% respectively. CypA knockdown resulted in an augmentation of cell populations in G0/G1 phase (M1) accompanied with a decrease in S & G2/M phase (M2 & M3). Cell proliferation decreased obviously in CypA knockdown BGC-823 cells, and cell cycle arrested in G0/G1 phase (M1). These results establish the relevance of CypA to gastric carcinoma proliferation in vitro. Conclusion Our results establish the relevance of CypA to gastric carcinoma proliferation in vitro. In further study we will do more experiments to reveal mechanisms how CypA plays roles in occurrence and development of gastric carcinoma and the pathways though which the signals of CypA transfer.