核糖核酸酶抑制因子与整合素连接激酶相互作用通过ILK/AKT/mTOR通路抑制膀胱癌体内外生长

doi:10.19401/j.cnki.1007-3639.2018.01.004

中国癌症杂志 ›› 2018, Vol. 28 ›› Issue (1): 30-37.doi: 10.19401/j.cnki.1007-3639.2018.01.004

核糖核酸酶抑制因子与整合素连接激酶相互作用通过ILK/AKT/mTOR通路抑制膀胱癌体内外生长

邢　雷¹，庄　翔²，陈俊霞²

1. 重庆医科大学附属第一医院内分泌乳腺外科，重庆 400016
2. 重庆医科大学细胞生物学与遗传学教研室，重庆 400016

出版日期:2018-01-30 发布日期:2018-02-07
通信作者: 陈俊霞　E-mail：chjunxia@126.com
基金资助:
国家自然科学基金(81672536，81372203，81172424)。

Ribonuclease inhibitor interacts with integrin-linked kinase and inhibits bladder cancer growth in vitro and in vivo through ILK/AKT/mTOR pathway

XING Lei¹, ZHUANG Xiang², CHEN Junxia²

1. Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; 2. Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China

Published:2018-01-30 Online:2018-02-07
Contact: CHEN Junxia E-mail: chjunxia@126.com

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摘要/Abstract

摘要： 背景与目的：蛋白质相互作用控制着细胞信号转导、跨膜转运、DNA合成及转录调控等生命过程，在生命活动中发挥重要作用。前期运用GST蛋白沉降技术与免疫共沉淀等已证明核糖核酸酶抑制因子(ribonuclease inhibitor，RI)与整合素连接激酶(intergrin-linked kinase，ILK)在体内外直接结合。该研究旨在探讨RI与ILK相互作用对ILK/AKT/mTOR通路与膀胱癌体内外生长的影响。方法：采用免疫荧光观察RI与ILK在膀胱癌EJ细胞中的共定位。采用荧光共振能量转移技术(fluorescence resonance energy transfer，FRET)验证RI与ILK的相互作用。构建过表达RI或ILK的EJ细胞系。采用蛋白［质］印迹法(Western blot)检测细胞中RI、ILK及ILK/AKT/mTOR通路相关蛋白的表达。采用细胞计数试剂盒(cell counting kit-8，CCK-8)与流式细胞术分析细胞增殖与细胞周期。构建膀胱癌裸鼠移植瘤模型，观察过表达RI或ILK对移植瘤生长的影响。采用免疫组织化学与免疫荧光分析瘤组织中RI、ILK及ILK/AKT/mTOR通路相关蛋白的表达。结果：EJ细胞中RI与ILK存在共定位的现象且RI与ILK存在相互作用。过表达RI抑制EJ细胞增殖并导致细胞周期阻滞于S期(P<0.05)，阻碍膀胱癌移植瘤的生长(P<0.05)，并抑制体内外ILK/AKT/mTOR通路的活化(P<0.05)。而过表达ILK促进细胞增殖与移植瘤的生长(P<0.05)，促进体内外ILK/AKT/mTOR信号通路的激活(P<0.05)。结论：RI通过与ILK相互作用阻碍ILK/AKT/mTOR通路激活并抑制膀胱癌体内外的增殖生长。

关键词: 核糖核酸酶抑制因子, 整合素连接激酶, 膀胱癌, 增殖

Abstract: Background and purpose: Protein interactions dominate the life processes, including cell signal transduction, transmembrane transport, DNA synthesis and transcriptional regulation, playing important and complex roles in life activities. Our previous study demonstrated the direct combination of ribonuclease inhibitor (RI) and integrin-linked kinase (ILK) in vitro and in vivo using GST pull down and Co-IP. The purpose of this study was to investigate the role of interplay between RI and ILK on ILK/AKT/mTOR pathway and bladder carcinoma growth in vitro and in vivo. Methods: Immunofluorescence staining was performed to analyze the co-localization of RI and ILK in bladder cancer EJ cells. Fluorescence resonance energy transfer (FRET) was applied to demonstrate the interaction between RI and ILK. Subsequently, the stable EJ cell lines with overexpression of RI or ILK were generated respectively. The protein levels of RI, ILK, and the molecules related to ILK/AKT/mTOR pathway were determined using Western blot. And the cell viability and cell cycle were analyzed by cell counting kit-8 (CCK-8) and flow cytometry assays respectively. In addition, the model of bladder cancer xenograft in nude mouse was constructed, and the effects of overexpression of RI or ILK on xenograft growth were observed. The expressions of RI, ILK and the proteins related to ILK/AKT/mTOR pathway in xenograft tissues were examined using immunohistochemistry and immunofluorescence. Results: The co-localization and interaction of RI and ILK were observed and verified in EJ cells. Overexpression of RI suppressed cell proliferation capacity (P<0.05), led to the blockage of S phase (P<0.05), repressed bladder cancer xenograft growth (P<0.05), and inhibited ILK/AKT/mTOR pathway in vitro and in vivo (P<0.05). However, overexpression of ILK promoted cell proliferation and xenograft growth (P<0.05), and overactivated ILK/AKT/mTOR pathway in vitro and in vivo (P<0.05). Conclusion: RI interacts with ILK, inhibits ILK/AKT/mTOR pathway and suppresses bladder cancer growth in vitro and in vivo.

Key words: Ribonuclease inhibitor, Integrin-linked kinase, Bladder cancer, Proliferation

邢　雷,庄　翔,陈俊霞. 核糖核酸酶抑制因子与整合素连接激酶相互作用通过ILK/AKT/mTOR通路抑制膀胱癌体内外生长[J]. 中国癌症杂志, 2018, 28(1): 30-37.

XING Lei, ZHUANG Xiang, CHEN Junxia. Ribonuclease inhibitor interacts with integrin-linked kinase and inhibits bladder cancer growth in vitro and in vivo through ILK/AKT/mTOR pathway[J]. China Oncology, 2018, 28(1): 30-37.

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核糖核酸酶抑制因子与整合素连接激酶相互作用通过ILK/AKT/mTOR通路抑制膀胱癌体内外生长

Ribonuclease inhibitor interacts with integrin-linked kinase and inhibits bladder cancer growth in vitro and in vivo through ILK/AKT/mTOR pathway

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