上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2018, Vol. 38 ›› Issue (2): 161-.doi: 10.3969/j.issn.1674-8115.2018.02.009

• 论著（基础研究） • 上一篇    下一篇

腺病毒介导的 siRNA 沉默PNUTS 基因对喉癌 Hep-2 细胞增殖、侵袭的影响

刘霞，余维，余丹，陈娟，骆小华，李兵   

  1. 重庆医科大学附属第一医院耳鼻喉头颈外科，重庆 400016
  • 出版日期:2018-02-28 发布日期:2018-03-09
  • 通讯作者: 李 兵，电子信箱：dclibing@sina.com。
  • 基金资助:
     重庆市自然科学基金（cstc2013jcyj A10059）

Effects of adenoviral-mediated siRNA targeting PNUTS on proliferation and invasion of laryngeal squamous cell carcinomas Hep-2 cells

LIU Xia, YU Wei, YU Dan, CHEN Juan, LUO Xiao-hua, LI Bing   

  1. Department of Otolaryngology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Online:2018-02-28 Published:2018-03-09
  • Supported by:
    Chongqing Natural Science Foundation, cstc2013jcyj A10059

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摘要： 目的 · 探讨腺病毒介导小 RNA 干扰（small interfering RNA，siRNA）下调蛋白磷酸酶 1 核目标亚基（phosphatase nuclear targeting
subunit，PNUTS）的表达对喉癌细胞 Hep-2 增殖、侵袭、迁移和上皮细胞间质化（epithelial-mesenchymal transition，EMT）的影响及机
制。方法 · 用携带表达 PNUTS siRNA 序列的重组腺病毒感染喉癌 Hep-2 细胞。实验分为空白对照（PBS）组、腺病毒空载（Ad-GFP）
组和实验（Ad-siPNUTS）组。实时荧光定量 PCR 及 Western blotting 检测细胞中 PNUTS 的表达。MTT 法检测细胞增殖情况。Transwell
法检测细胞侵袭、迁移能力。Western blotting 检测细胞中总 Rb、磷酸化 Rb（p-Rb）、PI3K、磷酸化 AKT（p-AKT）、E2F1、E-cadherin、
N-cadherin 和 ZEB1 蛋白的表达。结果 · 与 Ad-GFP 组比较，Ad-siPNUTS 组 Hep-2 细胞中 PNUTS mRNA 和蛋白（均 P=0.000）表达水平
降低；细胞增殖在第 2 日（P=0.004）、第 3 日（P=0.001）、第 4 日（P=0.000）受到抑制；细胞侵袭及迁移（均 P=0.000）能力降低；总
Rb（P=0.000）、p-Rb（P=0.000）、PI3K（P=0.023）、p-AKT（P=0.000）、E2F1（P=0.000）、N-cadherin（P=0.005）、ZEB1（P=0.000）
蛋白表达水平降低，E-cadherin（P=0.003）蛋白表达水平增加。结论 · Ad-siPNUTS 抑制喉癌细胞 Hep-2 的增殖、侵袭及转移能力，并逆
转 EMT，其可能与 PI3K/AKT 信号通路、Rb 信号通路有关。

关键词: 喉癌, 磷酸酶 1 核目标亚基, 磷酸化 Rb, 侵袭, 上皮细胞间质化

Abstract:

Objective · To explore the effects of adenovirus vector-mediated small interfering RNA (siRNA) targeting phosphatase nuclear targeting subunit
(PNUTS) on proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of laryngeal squamous cell carcinomas Hep-2 cells and its
mechanism. Methods · Recombinant adenovirus vector expressing PNUTS siRNA was infected into laryngeal squamous cell carcinomas Hep-2 cells and the
experiment was designed into PBS group, Ad-GFP group and Ad-siPNUTS group. Levels of PNUTS mRNA and protein were detected by real-time PCR and
Western blotting respectively. MTT assay was used to detect proliferation abilities of Hep-2 cells. Transwell assays were used to detect invasion and migration abilities of Hep-2 cells. The expression levels of total Rb, phosphorylated Rb (p-Rb), PI3K, phosphorylated AKT (p-AKT), E2F1, E-cadherin, N-cadherin and ZEB1 protein were detected by Western blotting. Results · Compared with Ad-GFP group, in Ad-siPNUTS group, the PNUTS mRNA and protein (both P=0.000) levels were dramatically decreased. The proliferation of Ad-siPNUTS infected Hep-2 cells were inhibited on the second day (P=0.004), the third day (P=0.001) and the fourth day (P=0.000). Meanwhile, the invasion and migration abilities of Ad-siPNUTS infected Hep-2 cells were decreased (both P=0.000). The expression levels of total Rb (P=0.000), p-Rb (P=0.000), PI3K (P=0.023), p-AKT (P=0.000), E2F1 (P=0.000), N-cadherin (P=0.005) and ZEB1 (P=0.000) were decreased while the E-cadherin (P=0.003) was increased. Conclusion · Ad-siPNUTS could inhibit the proliferation, invasion and migration abilities of Hep-2 cells and reverse the development of EMT, which may be related to PI3K/AKT signaling pathway and Rb signaling pathway.

Key words: laryngeal squamous cell carcinomas, phosphatase nuclear targeting subunit, phosphorylated Rb, invasion, epithelial-mesenchymal transition