Promoted cancer growth by stimulating cell proliferation and decreasing apoptosis using a lentivirus-based EphB2 RNAi in pancreatic carcinoma CFPAC-1 cells

Several studies have reported the change of EphB2 in a variety of carcinomas and suggested a functional relation between EphB2 and tumor progression. However, its role in human pancreatic carcinoma has not been described. The aim of this study was to evaluate the significance of EphB2 in human pancreatic carcinoma CFPAC-1 cells. A lentivirus-based RNA interference (RNAi) vector was designed, synthesized and transfected into CFPAC-1 cells to inhibit EphB2 expression. WST-8 based Colorimetric Assay Cell Counting kit 8 (CCK-8) in vitro and xenograft transplantation model in nude mice was used to evaluate cell proliferation and growth respectively. Cell-cycle and apoptosis were analyzed by flow cytometry (FCM). RT-PCR and Western blot were used to assess mRNA expression and protein levels. EphB2 expression was significantly suppressed both in mRNA and protein levels using the lentivirus-based EphB2 RNAi in CFPAC-1 cells (P < 0.01, P < 0.01). Silencing EphB2 stimulated cell growth in vitro (P < 0.05) and proliferation in vivo (P < 0.01) versus Control RNAi. EphB2 RNAi significantly increased S phase cells from 18.15 to 27.18% (P < 0.05), and significantly decreased G1 phase cells from 72.93 to 57.61% compared with Control RNAi (P < 0.05). In addition, decreased apoptosis was observed in CFPAC-1 EphB2 RNAi cells compared with Control RNAi cells (P < 0.01). The apoptosis rate was 1.63% and 7.44%, respectively. Silencing EphB2 increased CyclinD1, cyclindependent kinase 6 (CDK6) and Bcl-2 expression in both mRNA and protein levels compared with Control RNAi. A lentivirus-based EphB2 RNAi efficiently inhibited EphB2 gene and its protein expression. Silencing EphB2 stimulated pancreatic carcinoma growth by increasing cell proliferation through G1/S phase breakthrough, which relied on a CyclinD1/CDK6 cell-cycle regulated signal. Similarly, EphB2 inhibition also reduced CFPAC-1 cells apoptosis by upregulating Bcl-2 expression. Thus, at least in the context of pancreatic carcinoma CFPAC-1 cells, EphB2 plays a tumor suppressor role in cell proliferation and apoptosis. (C) 2011 Elsevier Masson SAS. All rights reserved.