Department of Integrative Hepatobiliary and Pancreatic Oncology, Fudan University Shanghai Cancer Center, 270 Dong An Road, Shanghai 200032, PR China.
Biomed Pharmacother. 2011 Mar;65(2):123-31. doi: 10.1016/j.biopha.2010.12.011. Epub 2011 Jan 21.
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 up-regulating 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.
已有多项研究报告 EphB2 在多种癌中的变化，并提示 EphB2 与肿瘤进展之间存在功能关系。然而，其在人胰腺癌细胞中的作用尚未描述。本研究旨在评估 EphB2 在人胰腺癌细胞 CFPAC-1 中的意义。设计、合成并转染慢病毒 EphB2 RNAi 载体至 CFPAC-1 细胞以抑制 EphB2 表达。体外 WST-8 比色细胞计数试剂盒 8 (CCK-8) 和裸鼠异种移植模型分别用于评估细胞增殖和生长。通过流式细胞术 (FCM) 分析细胞周期和凋亡。RT-PCR 和 Western blot 用于评估 mRNA 表达和蛋白水平。在 CFPAC-1 细胞中使用慢病毒 EphB2 RNAi 可显著抑制 EphB2 在 mRNA 和蛋白水平上的表达 (P<0.01, P<0.01)。与对照 RNAi 相比，沉默 EphB2 刺激细胞体外生长 (P<0.05) 和体内增殖 (P<0.01)。EphB2 RNAi 使 S 期细胞从 18.15%增加到 27.18% (P<0.05)，G1 期细胞从 72.93%减少到 57.61%，与对照 RNAi 相比 (P<0.05)。此外，与对照 RNAi 细胞相比，CFPAC-1 EphB2 RNAi 细胞中凋亡减少 (P<0.01)。凋亡率分别为 1.63%和 7.44%。沉默 EphB2 使 CyclinD1、细胞周期蛋白依赖性激酶 6 (CDK6) 和 Bcl-2 的 mRNA 和蛋白水平表达均增加，与对照 RNAi 相比。基于慢病毒的 EphB2 RNAi 可有效抑制 EphB2 基因及其蛋白表达。沉默 EphB2 通过 G1/S 期突破促进胰腺癌细胞增殖，从而依赖于细胞周期蛋白 D1/细胞周期蛋白依赖性激酶 6 细胞周期调控信号。同样，EphB2 抑制也通过上调 Bcl-2 表达减少 CFPAC-1 细胞凋亡。因此，至少在胰腺癌细胞 CFPAC-1 中，EphB2 在细胞增殖和凋亡中发挥肿瘤抑制作用。
