Zhai Hui-Hong, Meng Juan, Wang Jing-Bo, Liu Zhen-Xiong, Li Yuan-Fei, Feng Shan-Shan
Hui-Hong Zhai, Juan Meng, Department of Digestive Diseases, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China.
World J Gastroenterol. 2014 Aug 7;20(29):10062-70. doi: 10.3748/wjg.v20.i29.10062.
To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis.
The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed.
CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225).
CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.
探讨钙周期蛋白结合蛋白（也称为Siah-1相互作用蛋白，CacyBP/SIP）的核转位在胃癌发生中的作用。
采用蛋白质免疫印迹法检测胃癌细胞系中CacyBP/SIP蛋白的表达。对未受刺激或经胃泌素刺激的胃癌细胞系进行免疫荧光实验。为证实免疫荧光结果，通过蛋白质免疫印迹法评估CacyBP/SIP在细胞核和细胞质区室中的相对丰度。采用MTT法检测CacyBP/SIP核转位对细胞增殖的影响。采用集落形成实验检测克隆形成细胞的存活率。研究CacyBP/SIP核转位对细胞周期进程的影响。设计并构建两种CacyBP/SIP特异性小干扰RNA（siRNA）载体，以抑制CacyBP/SIP表达，从而减少CacyBP/SIP的核转位，并通过蛋白质免疫印迹法测定稳定转染细胞中CacyBP/SIP表达。随后评估抑制CacyBP/SIP核转位对细胞增殖产生的影响。
大多数胃癌细胞系中均存在CacyBP/SIP蛋白。未受刺激的细胞中，CacyBP/SIP分布于整个细胞质；而在受刺激的细胞中，CacyBP/SIP主要位于核周区域。CacyBP/SIP核转位对细胞产生生长刺激作用。CacyBP/SIP核转位组的集落数显著高于对照组。受刺激细胞中G1期的百分比显著低于对照细胞（对照细胞和经胃泌素处理的SGC7901细胞分别为69.70%±0.46%和65.80%±0.60%；P = 0.008；对照细胞和经胃泌素处理的MKN45细胞分别为72.99%±0.46%和69.36%±0.51%；P = 0.022）。CacyBP/SIPsi1有效下调CacyBP/SIP的表达，随后选择经CacyBP/SIPsi1稳定转染的细胞进行进一步细胞实验。在经CacyBP/SIPsi1稳定转染的细胞中，无论是否受刺激，CacyBP/SIP均分布于整个细胞质。如MTT法所示，CacyBP/SIP核转位减少后，对细胞增殖无显著影响。平板集落形成实验表明，刺激后锚定依赖性生长未增强。两种细胞系中G0-G1期细胞的百分比均无变化（对照细胞和经胃泌素处理的SGC7901-CacyBP/SIPsi1细胞分别为71.09%±0.16%和70.86%±0.25%；P = 0.101；对照细胞和经胃泌素处理的MKN45-CacyBP/SIPsi1细胞分别为74.17%±1.04%和73.07%±1.00%；P = 0.225）。
CacyBP/SIP核转位促进胃癌细胞的增殖和细胞周期进程。
