解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (4 ): 492-497.doi: 10.3969/j.issn.0529-1356.2013.04.009

• 肿瘤生物学 • 上一篇    下一篇

ZAC基因在奥曲肽抑制胃癌细胞体外增殖通路的作用

代稳1,2 丁一1 张钦宪1 *   

  1. 1.郑州大学基础医学院组织学胚胎学教研室，河南省分子医学重点学科开放实验室，郑州 450052; 2.解放军第一五三中心医院检验科，郑州 450042
  • 收稿日期:2012-09-13 修回日期:2012-12-18 出版日期:2013-08-06 发布日期:2013-09-04
  • 通讯作者: 张钦宪 E-mail:qxz53@zzu.edu.cn
  • 基金资助:

    省自然科学基金资助项目

Role of ZAC gene in the pathway of octreotide inhibiting proliferation of gastric cancer cells in vitro

DAI Wen 1,2 DING Yi1 ZHANG Qin-xian 1*   

  1. 1. Department of Histology and Embryology, College of Basic Science of Medicine, Zhengzhou University, Henan Key Laborary of Molecular Medicine, Zhengzhou 450052, China; 2. Clinical Laboratory，the 153rd Central Hospital of PLA Ji'nan Military Region, Zhengzhou 450042, China
  • Received:2012-09-13 Revised:2012-12-18 Online:2013-08-06 Published:2013-09-04

摘要：

目的 探讨ZAC基因在生长抑素类似物奥曲肽（OCT）抑制胃癌细胞增殖通路的作用。方法 分别以不同浓度OCT处理胃癌细胞BGC823和 SGC7901不同时间，MTT法筛选其增殖抑制的有效条件。OCT处理胃癌细胞（有效浓度/不同时间，有效时间/不同浓度），Western blotting检测
OCT对胃癌细胞ZAC基因的效应。设计3条ZAC基因RNA干扰片段，分别插入pSUPER-EGFP-I载体，构建3个ZAC-shRNA表达载体（pSUPER-EGFP-ZAC/1 pSUPER-EGFP-ZAC/2和pSUPER-EGFP-ZAC/3）。经酶切和序列分析鉴定后，分别转染胃癌细胞BGC823和SGC7901。经G418筛选，RT-PCR鉴定，建立
ZAC基因敲低（knock-down）的胃癌细胞系。以有效条件OCT孵育胃癌细胞（对照组）和ZAC基因敲低胃癌细胞（实验组），MTT法检测OCT对胃癌 细胞的生长抑制效应。结果 OCT抑制胃癌细胞增殖的有效条件为10nmol/L 孵育24h；OCT以时间和浓度依赖的方式诱导胃癌细胞ZAC基因表达。酶切及序列分析鉴定表明ZAC-shRNA表达载体构建成功。转染shRNA-ZAC/2的胃癌细胞，其ZAC mRNA水平明显降低（P<0.05），为ZAC基因敲低胃癌 细胞。ZAC基因敲低胃癌细胞的增殖明显高于对应的BGC823细胞和SGC7901细胞（P<0.05）。OCT孵育后，BGC823细胞和SGC7901细胞的增殖明显降低（P<0.05）。然而，ZAC基因敲低胃癌细胞的增殖无明显改变（P>0.05）。结论 OCT以时间和浓度依赖的方式诱导胃癌细胞ZAC基因表达；ZAC 基因在OCT抑制胃癌细胞增殖通路中具有重要作用。

关键词: 奥曲肽, ZAC, 胃癌细胞, 增殖, RNA干扰, 免疫印迹法

Abstract:

Objective To explore the role of ZAC gene in the pathway of somatostatin analogue, octreotide (OCT), inhibiting proliferation of gastric cancer cells. Methods The gastric cancer cells BGC823 and SGC7901 were treated with OCT at various concentrations for various times, respectively. MTT assay was used to screen effective condition of OCT for its antiproliferative action. After treatment with OCT (effective concentration/various times, effective time/various concentrations), the inducing effect of OCT on ZAC gene expression in gastric cancer cells was detected by Western blotting. Three fragments for ZAC gene RNA interference were inserted into pSUPER-EGFP-I vector to construct ZAC-shRNA expression vectors (pSUPER-EGFP-ZAC/1, pSUPER-EGFP-ZAC/2 and pSUPER-EGFP-ZAC/3), respectively. After identification by Hind Ⅲ/EcoRⅠdigestion and sequencing, the three ZAC-shRNA expression vectors were transfected into gastric cancer cells BGC823 and SGC7901, respectively. After G418 screening and RT-PCR identification, the gastric cancer cell lines in which ZAC gene was knock-down were established. Gastric cancer cells (control group) and those in which ZAC gene was knock-down (experimental group) were incubated with OCT at effective condition, the antiproliferative action of OCT was detected by MTT assay. Results The effective condition of OCT inhibiting proliferation of gastric cancer cells was 10nmol/L and treatmented for 24 hours. OCT induced ZAC gene expression in gastric cancer cells in a time-and dose-dependant manner. The results of Hind Ⅲ/EcoRⅠ digestion and sequencing indicated that ZAC-shRNA expression vectors were constructed successfully. The ZAC mRNA level in the gastric cancer cells transfected with shRNA-ZAC/2 was decreased significantly (P<0.05), which were the gastric cancer cell lines in which ZAC gene was knock-down. The proliferation of the gastric cancer cell lines in which ZAC gene was knock-down was significantly higher than that of corresponding BGC823 and SGC7901 (P<0.05). After OCT incubation, the proliferation of BGC823 and SGC7901 were decreased obviously (P<0.05); however, that of the cells in which ZAC gene was knock-down did not exhibit significant changes (P>0.05). Conclusion OCT induces ZAC gene expression in gastric cancer cells in a time-and dose-dependant manner. ZAC gene may play an important role in the pathway of OCT inhibiting the proliferation of gastric cancer cells.

Key words: Octreotide, ZAC, Gastric cancer cell, Proliferation, RNA interference, Western blotting