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
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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/m TOR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/m TOR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/m TOR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/m TOR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/m TOR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/m TOR通路激活并抑制膀胱癌体内外的增殖生长。
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/m TOR 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/m TOR 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/m TOR 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/m TOR 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/m TOR pathway in vitro and in vivo(P<0.05). Conclusion: RI interacts with ILK, inhibits ILK/AKT/m TOR pathway and suppresses bladder cancer growth in vitro and in vivo.
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/m TOR 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/m TOR 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/m TOR 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/m TOR 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/m TOR pathway in vitro and in vivo(P<0.05). Conclusion: RI interacts with ILK, inhibits ILK/AKT/m TOR pathway and suppresses bladder cancer growth in vitro and in vivo.
引文
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[11]FURIA A,MOSCATOM,CALìG,et al.The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei[J].FEBS Lett,2011,585(4):613-617.
[12]LALONDE S,EHRHARDT D W,LOQUéD,et al.Molecular and cellular approaches for the detection of protein-protein interactions:latest techniques and current limitations[J].Plant J,2008,53(4):610-635.
[13]LOU X Y,SONG N,YANG Y W.Fluorescence resonance energy transfer systems in supramolecular macrocyc Lnc chemistry[J].Molecules,2017,22(10):E1640.
[14]SHIRLEY L A,MCCARTY S,YANG M C,et al.Integrinlinked kinase affects signaling pathways and migration in thyroid cancer cells and is a potential therapeutic target[J].Surgery,2016,159(1):163-170.
[15]SATHE A,NAWROTH R.Targeting the PI3K/AKT/m TOR pathway in bladder cancer[J].Methods Mol Biol,2018,1655:335-350.
[16]ALTOMARE D A,KHALEDA R.Homeostasis and the importance for a balance between AKT/m TOR activity and intracellular signaling[J].Curr Med Chem,2012,19(22):3748-3762.
[17]PONTIER S M,HUCK L,WHITE D E,et al.Integrinlinked kinase has a critical role in Erb B2 mammary tumor progression:imp Lncations for human breast cancer[J].Oncogene,2010,29(22):3374-3385.
[18]PAN X,XIONG D,YAO X,et al.Up-regulating ribonuclease inhibitor inhibited epithelial-to-mesenchymal transition and metastasis in murine melanoma cells[J].Int J Biochem Cell Biol,2012,44(6):998-1008.
[19]XIONG D,LIOU Y,SHU J,et al.Down-regulating ribonuclease inhibitor enhances metastasis of bladder cancer cells through regulating epithelial-mesenchymal transition and ILK signaling pathway[J].Exp Mol Pathol,2014,96(3):411-421.
[2]FURIA A,MOSCATO M,CALìG,et al.The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei[J].FEBS Lett,2011,585(4):613-617.
[3]庄翔,张潞渝,吕梦欣,等.核糖核酸酶抑制因子与整合素连接激酶的相互作用以及对体外血管生成的影响[J].基础医学与临床,2016,36(8):1074-1079.
[4]BUITENHUIS M,VAN DER LINDEN E,ULFMAN L H,et al.Protein kinase B(PKB/c-akt)regulates homing of hematopoietic progenitors through modulation of their adhesive and migratory properties[J].Blood,2010,116(13):2373-2384.
[5]MCDONALD P C,FIELDING A B,DEDHAR S.Integrinlinked kinase--essential roles in physiology and cancer biology[J].J Cell Sci,2008,121(Pt 19):3121-3132.
[6]QU Y,HAO C,XU J,et al.ILK promotes cell proliferation in breast cancer cells by activating the PI3K/Akt pathway[J].Mol Med Rep,2017,16(4):5036-5042.
[7]庄翔,朱军,吕梦欣,等.特异性ILK si RNA对膀胱癌EJ细胞EMT及移植瘤生长的影响[J].基础医学与临床,2016,36(2):191-198.
[8]李林,舒静,刘玉林,等.核糖核酸酶抑制因子与血管生成素的相互作用[J].第三军医大学学报,2014,36(14):1454-1458.
[9]JOHNSON R J,MCCOY J G,BINGMAN C A,et al.Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein[J].J Mol Biol,2007,368(2):434-449.
[10]PIZZO E,SARCINELLI C,SHENG J,et al.Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival[J].J Cell Sci,2013,126(Pt 18):4308-4319.
[11]FURIA A,MOSCATOM,CALìG,et al.The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei[J].FEBS Lett,2011,585(4):613-617.
[12]LALONDE S,EHRHARDT D W,LOQUéD,et al.Molecular and cellular approaches for the detection of protein-protein interactions:latest techniques and current limitations[J].Plant J,2008,53(4):610-635.
[13]LOU X Y,SONG N,YANG Y W.Fluorescence resonance energy transfer systems in supramolecular macrocyc Lnc chemistry[J].Molecules,2017,22(10):E1640.
[14]SHIRLEY L A,MCCARTY S,YANG M C,et al.Integrinlinked kinase affects signaling pathways and migration in thyroid cancer cells and is a potential therapeutic target[J].Surgery,2016,159(1):163-170.
[15]SATHE A,NAWROTH R.Targeting the PI3K/AKT/m TOR pathway in bladder cancer[J].Methods Mol Biol,2018,1655:335-350.
[16]ALTOMARE D A,KHALEDA R.Homeostasis and the importance for a balance between AKT/m TOR activity and intracellular signaling[J].Curr Med Chem,2012,19(22):3748-3762.
[17]PONTIER S M,HUCK L,WHITE D E,et al.Integrinlinked kinase has a critical role in Erb B2 mammary tumor progression:imp Lncations for human breast cancer[J].Oncogene,2010,29(22):3374-3385.
[18]PAN X,XIONG D,YAO X,et al.Up-regulating ribonuclease inhibitor inhibited epithelial-to-mesenchymal transition and metastasis in murine melanoma cells[J].Int J Biochem Cell Biol,2012,44(6):998-1008.
[19]XIONG D,LIOU Y,SHU J,et al.Down-regulating ribonuclease inhibitor enhances metastasis of bladder cancer cells through regulating epithelial-mesenchymal transition and ILK signaling pathway[J].Exp Mol Pathol,2014,96(3):411-421.