胃泌素调控ERK信号通路在促进大肠癌CACO2细胞增殖中的作用

中国临床药理学与治疗学 ›› 2017, Vol. 22 ›› Issue (4): 401-405.

胃泌素调控ERK信号通路在促进大肠癌CACO2细胞增殖中的作用

茆家定，胡迪，吴佩

皖南医学院弋矶山医院胃肠外科，芜湖 241001，安徽

收稿日期:2017-03-07 修回日期:2017-03-23 出版日期:2017-04-26 发布日期:2017-04-26
作者简介:茆家定，男，博士，主任医师，硕士生导师，研究方向：胃肠激素与消化道肿瘤。 E-mail: maojiading0205@sina.com
基金资助:
安徽省自然科学基金资助项目（1408085MH148）；皖南医学院弋矶山医院人才引进基金（YR201406）

Effects of gastrin on ERK signaling pathway in the proliferation of colorectal cancer cell line CACO2

MAO Jiading, HU Di, WU Pei

Department of Gastrointestinal Surgery, Yijishan Hospital,the First Affiliated Hospital of Wannan Medical College, Wuhu 241001, Anhui, China

Received:2017-03-07 Revised:2017-03-23 Online:2017-04-26 Published:2017-04-26

摘要/Abstract

摘要：

目的: 利用RNA干扰技术探讨ERK1/2基因在胃泌素促进大肠癌细胞株CACO2增殖中的作用及分子机制。方法: 通过慢病毒感染细胞构建胃泌素受体（CCK-BR）阳性的细胞稳转株CACO2，应用qRT-PCR检测大肠癌细胞株CACO2中CCK-BR的表达情况。应用RNA干扰技术沉默ERK1/2基因后，分析其总的ERK基因的蛋白表达及其磷酸化水平变化。实验共分4组：对照组、阴性干扰组、胃泌素组及质粒干扰组。使用流式细胞仪Annexin V-FITC法检测各组细胞增殖指数及Western blot检测各组细胞ERK1/2蛋白的表达和磷酸化水平。结果: 慢病毒感染后,在CACO2细胞中均检测到目的条带膜蛋白，CACO2细胞存在着一定量的CCK-BR mRNA表达，扩增产物为185 bp。筛选出干扰质粒，质粒与脂质体比例在1∶2，转染效率达到40%～50%，转染干扰质粒的细胞中ERK蛋白表达水平降低，从蛋白水平说明构建的质粒沉默有效。胃泌素组细胞增殖指数明显高于质粒干扰组，也高于阴性干扰组和空白对照组，差异有统计学意义(P<0.01)。4组细胞间ERK1/2的蛋白表达及磷酸化水平比较，差异有统计学意义(P<0.01)。胃泌素组ERK1/2的蛋白表达及磷酸化水平明显高于质粒干扰组，也高于阴性干扰组和空白对照组，差异有统计学意义(P<0.01)。结论: 胃泌素能够通过ERK信号通路促进体外大肠癌CACO2细胞增殖。通过RNA干扰技术有效地阻断ERK信号通路下调ERK蛋白磷酸化水平，有望为胃泌素依赖型大肠癌综合治疗寻找到一条新的切入途径。

关键词: 胃泌素, ERK1/2, 大肠癌, RNA干扰, 慢病毒感染

Abstract:

AIM: To investigate the effects and mechanisms of exrtacellular-signal regulated protein kinase in gastrin-induced cell proliferation of colorectal cancer cells by using RNA interference technology. METHODS: CCK-BR positive cell strain CACO2 was constructed lentivirus infection. Detection of gastrin receptor (CCK-BR) expression in colorectal cancer cell line CACO2 by qRT-PCR. RNA interference was used to silence the ERK1/2 gene, and the expression of total ERK protein and its phosphorylation level were analyzed. The experiment was divided into four groups: The control group, negative interference group, gastrin group and plasmid interference group. Changes of proliferation index of CACO2 cells were detected by cell cytometry. The protein of ERKl/2 and phosphorylation levels were detected by Western blot. RESULTS: After chronic viral infection, the target strip membrane protein and mRNA expression of CCK-BR were detected in the CACO2 cells. The amplified product was 185 bp. The interference plasmid was selected, the ratio of plasmid and liposome was 1∶2, the transfection efficiency was 40%-50%, and the expression level of ERK protein in the transfected plasmid was decreased. From the protein level,the constructed plasmid was observed effective. The proliferation index of the gastrin group was significantly higher than that of the plasmid interference group,the negative interference group and the blank control group, the difference was statistically significant (P<0.01). The expression of ERK1/2 protein and phosphorylation level in the four groups were significantly different (P<0.01). The protein expression and phosphorylation level of ERK1/2 in gastrin group were significantly higher than those in the plasmid interference group, the negative interference group and the blank control group, the difference was statistically significant (P<0.01). CONCLUSION: Gastrin can promote the proliferation of CACO2 cells in vitro by ERK signaling pathway. RNA interference can effectively block the phosphorylation of ERK protein in ERK signaling pathway, which is expected to provide a new approach for the treatment of colorectal cancer.

Key words: gastrin, ERK1/2, colorectal cancer, RNAi, lentivirus infection

中图分类号:

R965.2
R73-3

茆家定，胡迪，吴佩. 胃泌素调控ERK信号通路在促进大肠癌CACO2细胞增殖中的作用[J]. 中国临床药理学与治疗学, 2017, 22(4): 401-405.

MAO Jiading, HU Di, WU Pei. Effects of gastrin on ERK signaling pathway in the proliferation of colorectal cancer cell line CACO2[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2017, 22(4): 401-405.

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