Department of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361004, P.R. China.
Mol Med Rep. 2013 Apr;7(4):1180-4. doi: 10.3892/mmr.2013.1342. Epub 2013 Feb 26.
The aim of this study was to investigate the effect of aplasia ras homolog member I (ARHI) on proliferation, apoptosis and the cell cycle in the pancreatic cancer cell line PANC-1. The study also aimed to examine the effect of ARHI on the activity of the nuclear factor (NF)-κB and to determine whether ARHI acts as a tumor suppressor in the development of pancreatic cancer by inhibiting the activity of NF-κB. A pIRES2‑EGFP‑ARHI vector, constructed by reverse transcrition (RT)‑PCR, was transiently transfected into the PANC-1 cells and analyzed for the expression of the ARHI protein by western blotting. A MTT assay was used to quantify cell proliferation, and apoptosis was analyzed by flow cytometry. The NF‑κB signaling pathway, specifically the pathway using the nuclear phosphorylated p65 isoform, was analyzed by western blotting. Expression of the ARHI protein was detected by western blotting subsequent to the PANC-1 cells being transiently transfected with the pIRES2‑EGFP‑ARHI construct. Cell proliferation was strongly inhibited in the PANC-1 cells transfected with pIRES2‑EGFP‑ARHI. The cell cycle assays indicated an increase in the number of cells at the G0/G1 phase and a decrease in the cells at the S phase, but the difference was not significant (P>0.05). Time course studies also indicated a marked increase in the apoptotic index following transient transfection, as well as a gradual decrease in the expression of the nuclear phosphorylated p65 protein. ARHI acts as a tumor suppressor by downregulating the NF‑κB signaling pathway, which results in the inhibition of cell proliferation, apoptosis and the cell cycle in the pancreatic tumor PANC-1 cell line.
本研究旨在探讨发育抑制基因 Ras 同源物 I（ARHI）对胰腺癌细胞系 PANC-1 增殖、凋亡和细胞周期的影响。本研究还旨在探讨 ARHI 对核因子（NF）-κB 活性的影响，并确定 ARHI 是否通过抑制 NF-κB 活性而作为胰腺癌发生的肿瘤抑制因子。通过逆转录（RT）-PCR 构建了 pIRES2-EGFP-ARHI 载体，将其瞬时转染到 PANC-1 细胞中，并通过 Western blot 分析 ARHI 蛋白的表达。MTT 法检测细胞增殖，流式细胞术检测细胞凋亡。Western blot 法分析 NF-κB 信号通路，特别是核磷酸化 p65 同工型的信号通路。转染 pIRES2-EGFP-ARHI 构建体后，通过 Western blot 检测 PANC-1 细胞中 ARHI 蛋白的表达。pIRES2-EGFP-ARHI 转染的 PANC-1 细胞增殖受到强烈抑制。细胞周期分析表明 G0/G1 期细胞数量增加，S 期细胞数量减少，但差异无统计学意义（P>0.05）。时程研究还表明，瞬时转染后细胞凋亡指数明显增加，核磷酸化 p65 蛋白表达逐渐下降。ARHI 通过下调 NF-κB 信号通路发挥肿瘤抑制作用，从而抑制胰腺肿瘤 PANC-1 细胞系的细胞增殖、凋亡和细胞周期。
