宫颈癌细胞中与UTF1相互作用的蛋白分析
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摘要
目的探讨宫颈癌细胞中与转录辅激活因子(UTF1)相互作用的蛋白。方法构建稳定表达FLAG-HA双标签标记的UTF1蛋白的SiHa细胞株,利用FLAG-HA串联亲和纯化(TAP)双标签纯化实验,对目的条带进行质谱分析。结果成功构建稳定表达FLAG-HA双标签标记的UTF1细胞株。通过质谱分析得到UTF1相互作用的蛋白数据,结果显示UTF1捕获蛋白参与DNA修复、代谢、核糖体、细胞连接、细胞因子相互作用、吞噬等多种生物学过程,以及Jak-STAT、MAPK、mTOR、VEGF、 wnt等多条信号通路,并与系统性红斑狼疮、帕金森症、阿尔兹海默症、自身免疫性疾病等的发病相关。结论 UTF1捕获蛋白参与细胞多种生理及病理过程,为进一步了解UTF1在肿瘤发病机制中的作用提供了新的线索。
Objective To search for the proteins interacting with undifferentiated embryonic cell transcription factor 1(UTF1) in cervical cancer cells. Methods We constructed SiHa cell line which stably expressed the UTF1 protein labeled with FLAG-HA double tag. After FLAG-HA double-tag tandem affinity purification experiment. The protein composition of target band was determined using mass-spectrometry. Results The UTF1 cell line that stably expressed FLAG-HA double-tag was successfully constructed. Through mass-spectrometry analysis, we found that captured protein of UTF1 is involved in various biological processes such as DNA repair, metabolism, ribosome, cell conjunction, interacions among various cytokines, phagocytosis, and many signal pathways including Jak-STAT, MAPK, mTOR, VEGF, wnt. The captured protein was associated with the development of systemic lupus erythematosus, Parkinson's disease, Alzheimer's disease, and autoimmune diseases. Conclusion The captured protein of UTF1 is involved in a variety of physiological and pathological processes of the cells, providing new clues for further understanding of the role of UTF1 in the pathogenesis of tumors.
Objective To search for the proteins interacting with undifferentiated embryonic cell transcription factor 1(UTF1) in cervical cancer cells. Methods We constructed SiHa cell line which stably expressed the UTF1 protein labeled with FLAG-HA double tag. After FLAG-HA double-tag tandem affinity purification experiment. The protein composition of target band was determined using mass-spectrometry. Results The UTF1 cell line that stably expressed FLAG-HA double-tag was successfully constructed. Through mass-spectrometry analysis, we found that captured protein of UTF1 is involved in various biological processes such as DNA repair, metabolism, ribosome, cell conjunction, interacions among various cytokines, phagocytosis, and many signal pathways including Jak-STAT, MAPK, mTOR, VEGF, wnt. The captured protein was associated with the development of systemic lupus erythematosus, Parkinson's disease, Alzheimer's disease, and autoimmune diseases. Conclusion The captured protein of UTF1 is involved in a variety of physiological and pathological processes of the cells, providing new clues for further understanding of the role of UTF1 in the pathogenesis of tumors.
引文
[1] Nishimoto M,Fukushima A,Okuda A,et al.The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2[J].Mol Cell Biol,1999,19(8):5453-5465.
[2] Nishimoto M,Miyagi S,Yamagishi T,et al.Oct-3/4 maintains the proliferative embryonic stem cell state via specific binding to a variant octamer sequence in the regulatory region of the UTF1 locus[J].Mol Cell Biol,2005,25(12):5084-5094.DOI:10.1128/MCB.25.12.5084-5094.2005.
[3] Kooistra S M,Thummer R P,Eggen B J.Characterization of human UTF1,a chromatin-associated protein with repressor activity expressed in pluripotent cells[J].Stem Cell Res,2009,2(3):211-218.DOI:10.1016/j.scr.2009.02.001.
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[5] Galonska C,Ziller M J,Karnik R,et al.Ground State Conditions Induce Rapid Reorganization of Core Pluripotency Factor Binding before Global Epigenetic Reprogramming[J].Cell Stem Cell,2015,17(4):462-470.DOI:10.1016/j.stem.2015.07.005.
[6] Herbst R A,Weiss J,Ehnis A,et al.Loss of heterozygosity for 10q22-10qter in malignant melanoma progression[J].Cancer Res,1994,54(12):3111-3114.
[7] Maier D,Comparone D,Taylor E,et al.New deletion in low-grade oligodendroglioma at the glioblastoma suppressor locus on chromosome 10q25-26[J].Oncogene,1997,15(8):997-1000.DOI:10.1038/sj.onc.1201209.
[8] Nagase S,Yamakawa H,Sato S,et al.Identification of a 790-kilobase region of common allelic loss in chromosome 10q25-q26 in human endometrial cancer[J].Cancer Res,1997,57(9):1630-1633.
[9] Wang P,Li J,Allan R W,et al.Expression of UTF1 in primary and metastatic testicular germ cell tumors[J].Am J ClinPathol,2010,134(4):604-612.
[10] Liu A,Cheng L,Du J,et al.Diagnostic utility of novel stem cell markers SALL4,OCT4,NANOG,SOX2,UTF1,and TCL1 in primary mediastinal germ cell tumors[J].Am J SurgPathol,2010,34(5):697-706.
[11] Xu C,Zhou Y,Chen W.Expression of undifferentiated embryonic cell transcription factor-1 (UTF1) in breast cancers and their matched normal tissues[J].Cancer Cell Int,2014,14(1):116.
[12] Wu X L,Zheng P S.Undifferentiated embryonic cell transcription factor-1 (UTF1) inhibits the growth of cervical cancer cells by transactivating p27Kip1[J].Carcinogenesis,2013,34(7):1660-1668.DOI:10.1093/carcin/bgt102.
[13] Guenin S,Mouallif M,Deplus R,et al.Aberrant promoter methylation and expression of UTF1 during cervical carcinogenesis[J].PLoS One,2012,7(8):e42704.
[14] Mouallif M,Albert A,Zeddou M,et al.Expression profile of undifferentiated cell transcription factor 1 in normal and cancerous human epithelia[J].Int J ExpPathol,2014,95(4):251-259.
[15] van den Boom V,Kooistra S M,Boesjes M,et al.UTF1 is a chromatin-associated protein involved in ES cell differentiation[J].J Cell Biol,2007,178(6):913-924.
[16] Kooistra S M,van den Boom V,Thummer R P,et al.Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression[J].Stem Cells,2010,28(10):1703-1714.DOI:10.1002/stem.497.
[17] Ramachandran I,Thavathiru E,Ramalingam S,et al.Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth,invasion and angiogenesis in vivo[J].Oncogene,2012,31(22):2725-2737.DOI:10.1038/onc.2011.455.
[18] Wei L H,Kuo M L,Chen C A,et al.Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway[J].Oncogene,2003,22(10):1517-1527.
[19] Tomkiel J E,Alansari H,Tang N,et al.Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer[J].Clin Cancer Res,2002,8(3):752-758.
[20] Givalos N,Gakiopoulou H,Skliri M,et al.Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer[J].Mod Pathol,2007,20(2):159-166.DOI:10.1038/modpathol.3800719.
[21] Levidou G,Gakiopoulou H,Kavantzas N,et al.Prognostic significance of replication protein A (RPA) expression levels in bladder urothelial carcinoma[J].BJU Int,2011,108( 2):59-65.
[22] Namkoong S,Lee E J,Jang I S,et al.Elevated level of human RPA interacting protein alpha (hRIPalpha) in cervical tumor cells is involved in cell proliferation through regulating RPA transport[J].FEBS Lett,2012,586(20):3753-3760.
[2] Nishimoto M,Miyagi S,Yamagishi T,et al.Oct-3/4 maintains the proliferative embryonic stem cell state via specific binding to a variant octamer sequence in the regulatory region of the UTF1 locus[J].Mol Cell Biol,2005,25(12):5084-5094.DOI:10.1128/MCB.25.12.5084-5094.2005.
[3] Kooistra S M,Thummer R P,Eggen B J.Characterization of human UTF1,a chromatin-associated protein with repressor activity expressed in pluripotent cells[J].Stem Cell Res,2009,2(3):211-218.DOI:10.1016/j.scr.2009.02.001.
[4] Kristensen D M,Nielsen J E,Skakkebaek N E,et al.Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms[J].Hum Reprod,2008,23(4):775-782.DOI:10.1093/humrep/den010.
[5] Galonska C,Ziller M J,Karnik R,et al.Ground State Conditions Induce Rapid Reorganization of Core Pluripotency Factor Binding before Global Epigenetic Reprogramming[J].Cell Stem Cell,2015,17(4):462-470.DOI:10.1016/j.stem.2015.07.005.
[6] Herbst R A,Weiss J,Ehnis A,et al.Loss of heterozygosity for 10q22-10qter in malignant melanoma progression[J].Cancer Res,1994,54(12):3111-3114.
[7] Maier D,Comparone D,Taylor E,et al.New deletion in low-grade oligodendroglioma at the glioblastoma suppressor locus on chromosome 10q25-26[J].Oncogene,1997,15(8):997-1000.DOI:10.1038/sj.onc.1201209.
[8] Nagase S,Yamakawa H,Sato S,et al.Identification of a 790-kilobase region of common allelic loss in chromosome 10q25-q26 in human endometrial cancer[J].Cancer Res,1997,57(9):1630-1633.
[9] Wang P,Li J,Allan R W,et al.Expression of UTF1 in primary and metastatic testicular germ cell tumors[J].Am J ClinPathol,2010,134(4):604-612.
[10] Liu A,Cheng L,Du J,et al.Diagnostic utility of novel stem cell markers SALL4,OCT4,NANOG,SOX2,UTF1,and TCL1 in primary mediastinal germ cell tumors[J].Am J SurgPathol,2010,34(5):697-706.
[11] Xu C,Zhou Y,Chen W.Expression of undifferentiated embryonic cell transcription factor-1 (UTF1) in breast cancers and their matched normal tissues[J].Cancer Cell Int,2014,14(1):116.
[12] Wu X L,Zheng P S.Undifferentiated embryonic cell transcription factor-1 (UTF1) inhibits the growth of cervical cancer cells by transactivating p27Kip1[J].Carcinogenesis,2013,34(7):1660-1668.DOI:10.1093/carcin/bgt102.
[13] Guenin S,Mouallif M,Deplus R,et al.Aberrant promoter methylation and expression of UTF1 during cervical carcinogenesis[J].PLoS One,2012,7(8):e42704.
[14] Mouallif M,Albert A,Zeddou M,et al.Expression profile of undifferentiated cell transcription factor 1 in normal and cancerous human epithelia[J].Int J ExpPathol,2014,95(4):251-259.
[15] van den Boom V,Kooistra S M,Boesjes M,et al.UTF1 is a chromatin-associated protein involved in ES cell differentiation[J].J Cell Biol,2007,178(6):913-924.
[16] Kooistra S M,van den Boom V,Thummer R P,et al.Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression[J].Stem Cells,2010,28(10):1703-1714.DOI:10.1002/stem.497.
[17] Ramachandran I,Thavathiru E,Ramalingam S,et al.Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth,invasion and angiogenesis in vivo[J].Oncogene,2012,31(22):2725-2737.DOI:10.1038/onc.2011.455.
[18] Wei L H,Kuo M L,Chen C A,et al.Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway[J].Oncogene,2003,22(10):1517-1527.
[19] Tomkiel J E,Alansari H,Tang N,et al.Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer[J].Clin Cancer Res,2002,8(3):752-758.
[20] Givalos N,Gakiopoulou H,Skliri M,et al.Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer[J].Mod Pathol,2007,20(2):159-166.DOI:10.1038/modpathol.3800719.
[21] Levidou G,Gakiopoulou H,Kavantzas N,et al.Prognostic significance of replication protein A (RPA) expression levels in bladder urothelial carcinoma[J].BJU Int,2011,108( 2):59-65.
[22] Namkoong S,Lee E J,Jang I S,et al.Elevated level of human RPA interacting protein alpha (hRIPalpha) in cervical tumor cells is involved in cell proliferation through regulating RPA transport[J].FEBS Lett,2012,586(20):3753-3760.
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