天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (6): 590-594.doi: 10.11958/j.issn.0253-9896.2015.06.004

• 细胞与分子生物学 • 上一篇    下一篇

siRNA干扰Gab1表达对胆管癌细胞株HUCCA-1功能的影响及其与PI3K/Akt信号通路的关系

姚旭， 田忠， 刘源   

  1. 中国医科大学附属盛京医院第十普外科

  • 收稿日期:2014-11-21 修回日期:2015-01-30 出版日期:2015-06-15 发布日期:2015-06-10
  • 通讯作者: 姚旭 E-mail:yxyaoxu@aliyun.com
  • 基金资助:
    辽宁省科技攻关计划项目 （2013408001）

Effect of silencing Gab1 expression on cholangiocarcinoma cell line HUCCA-1 and its#br# correlation with PI3K/Akt pathway

YAO Xu, TIAN Zhong, LIU Yuan   

  1. Department of the 10th General Surgery， Shengjing Hospital of China medical university

  • Received:2014-11-21 Revised:2015-01-30 Published:2015-06-15 Online:2015-06-10
  • Contact: Xu YAO E-mail:yxyaoxu@aliyun.com

PDF (PC)

4289

摘要： 目的 检测下调 Gab1 表达后人胆管癌细胞株 HUCCA-1 细胞功能、 PI3KCA 及 Akt1 的蛋白及 mRNA 表达水平改变， 探讨 Gab1、 PI3KCA 及 Akt1 表达在胆管癌恶性行为中的作用机制。方法 Gab1siRNA 转染 HUCCA-1细胞， 采用 qRT-PCR 法及 Western blot 法检测转染效率及 PI3KCA、 Akt1 表达水平， MTT 检测转染后细胞增殖变化，流式细胞术检测转染后细胞凋亡变化， Transwell 检测细胞迁移及侵袭能力。结果 Gab1siRNA 在 HUCCA-1 细胞中的转染效率约为 65%～70%， 转染效率佳； Gab1siRNA 转染 HUCCA-1 细胞后， PI3KCA、 Akt1 的蛋白及 mRNA 表达量下调； Gab1siRNA 组 HUCCA-1 细胞增殖能力在 48 h、 72 h 及 96 h 均低于 siRNA control 组和 Mock 组（P < 0.05）；Gab1siRNA 组的 HUCCA-1 细胞凋亡率高于 Mock 及 siRNA control 组 （P < 0.05）； Gab1siRNA 组 HUCCA-1 细胞迁移减少百分比及侵袭能力低于 siRNA control 组及 Mock 组（P＜0.05）。结论 Gab1 可能通过激活 PI3K/Akt 信号通路表达促进肿瘤细胞恶性行为，Gab1 可作为胆管癌治疗新的靶向标志物及候选基因。

关键词: 胆管癌, 增殖, 凋亡, Gab1, HUCCA-1, PI3K/Akt 信号通路

Abstract: Objective To explore the functionalternation of human cholangiocarcinoma cell line HUCCA-1 by silencing Gab1 expression; to detect its effect on PI3KCA and Akt1 expression at protein and mRNA levels and to explore the roleof Gab1， PI3KCA and Akt1 expressions in the malignant behavior of cholangiocarcinoma. Methods Gab1 siRNA was trans⁃fected into cholangiocarcinoma cell line HUCCA-1. Proliferation, apoptosis, migration/ invasion of cells were examined byMTT, flow cytometry and Transwell assay respectively after transfection. PI3KCA and Akt1 expressions were detected by Western blotting and qPCR. Results Transfection efficiency was satisfactory and reaches 65%～70%； PI3KCA and Akt1 expressions were down- regulated at both protein and mRNA levels upon silencing of Gab1. Meanwhile, proliferations of HUCCA-1 cells were inhibited (P < 0.01） ;apoptosis rate increases（P < 0.05）; and migration and invasion were both inhibited upon Gab1 silencing（P < 0.05） . Conclusion The proliferation, migration and invasion are all inhibited while apoptosis is attenuated after Gab1 was down-regulated by Gab1 siRNA transfection. PI3KCA and Akt1 expressions are up-regulated
with down-regulation of Gab1. Therefore, Gab1 may enhance the maglinant behavior of cholangiocarcinoma cells through upregulating PI3KCA and Akt1 expression and it can be used as a candidate for cholangiocarcinoma therapy.



Key words: cholangiocarcinoma, proliferation, apoptosis, Gab1, HUCCA-1, PI3K/Akt pathway