宫颈癌HeLa细胞内SKP2结合蛋白的筛选和功能预测
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摘要
目的:通过免疫共沉淀与质谱分析技术从宫颈癌HeLa细胞中获取与S期激酶相关蛋白2(S-phase kinase-associated protein 2,SKP2)结合的蛋白质群体并预测其生物功能。方法:以免疫共沉淀与Western blotting技术建立SKP2免疫共沉淀体系,以SDS-PAGE和银染技术获得SKP2结合蛋白的特异条带,通过质谱分析技术获得可能与SKP2结合的蛋白群体,应用生物信息学技术对筛选得到的蛋白进行GO分析与KEGG分析。结果:HeLa细胞内存在一定水平的SKP2蛋白表达,可进行免疫共沉淀反应;成功建立了SKP2免疫共沉淀体系,并获得SKP2结合蛋白样品;针对差异的凝胶条带进行质谱分析,共鉴定出SKP2结合蛋白563个;设定筛选条件后,获得可信度较高的SKP2蛋白270个,进行GO分析与KEGG分析后初步预测了结合蛋白参与的细胞功能和信号通路。结论:从宫颈癌HeLa细胞中成功筛选获取SKP2结合蛋白,为后续筛选靶标结合蛋白和寻找细胞靶向药物奠定了基础。
Objective:The co-immunoprecipitation and mass spectrometric analysis was carried out to obtain the S-phase kinase-associated protein 2(SKP2)-binding proteins in HeLa cells, and the biological functions of these binding proteins were forecast. Methods:The co-immunoprecipitation system was established by co-immunoprecipitation and Western blotting assay; the specific protein gel of SKP2-binding proteins was obtained by SDS-PAGE and silver staining assay; the potential SKP2-binding proteins was identified by mass spectrometric analysis; and the GO(Gene ontology) analysis and KEGG analysis was carried out by bioinformatics technique.Results: The expression level of SKP2 protein in HeLa cells was high enough for co-immunoprecipitation assay; the co-immunoprecipitation system was established successfully, and SKP2-binding proteins was obtained; a total of 563 proteins were identified by mass spectrometric analysis, and 270 proteins with high credibility were obtained after screening. The GO analysis and KEGG analysis was carried out for the 270 proteins to forecast their functions and pathways. Conclusion: The SKP2-binding proteins were screened successfully, and it was the foundation for the subsequent screening of target-binding proteins and the search for targeting drugs.
Objective:The co-immunoprecipitation and mass spectrometric analysis was carried out to obtain the S-phase kinase-associated protein 2(SKP2)-binding proteins in HeLa cells, and the biological functions of these binding proteins were forecast. Methods:The co-immunoprecipitation system was established by co-immunoprecipitation and Western blotting assay; the specific protein gel of SKP2-binding proteins was obtained by SDS-PAGE and silver staining assay; the potential SKP2-binding proteins was identified by mass spectrometric analysis; and the GO(Gene ontology) analysis and KEGG analysis was carried out by bioinformatics technique.Results: The expression level of SKP2 protein in HeLa cells was high enough for co-immunoprecipitation assay; the co-immunoprecipitation system was established successfully, and SKP2-binding proteins was obtained; a total of 563 proteins were identified by mass spectrometric analysis, and 270 proteins with high credibility were obtained after screening. The GO analysis and KEGG analysis was carried out for the 270 proteins to forecast their functions and pathways. Conclusion: The SKP2-binding proteins were screened successfully, and it was the foundation for the subsequent screening of target-binding proteins and the search for targeting drugs.
引文
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[3]FU H C,YANG Y C,CHEN Y J,et.al.Increased expression of SKP2is an independent predictor of locoregional recurrence in cervical cancer via promoting DNA-damage response after irradiation[J].Oncotarget,2016,7(28):44047-44061.DOI:10.18632/oncotarget.10057.
[4]CHEN T P,CHEN C M,CHANG H W,et al.Increased expression of SKP2 and phospho-MAPK/ERK1/2 and decreased expression of p27during tumor progression of cervical neoplasms[J].Gynecologic Oncol,2007,104(3):516-523.DOI:10.1016/j.ygyno.2006.09.015.
[5]NARAYAN G,MURTY V V.Integrative genomic approaches in cervical cancer:implications for molecular pathogenesis[J].Future oncology,2010,6(10):1643-1652.DOI:10.2217/fon.10.114.
[6]HARRIS M A,CLARK J,IRELAND A,et al.The gene ontology(GO)database and informatics resource[J].Nucleic Acids Res,2004,32(suppl 1):D258-261.DOI:10.1093/nar/gkh036.
[7]KANEHISA M,GOTO S.KEGG:Kyoto encyclopedia of genes and genomes[J].Nucleic Acids Res,2000,28(1):27-30.DOI:10.7717/peerj.1991/supp-10.
[8]WANG G,CHAN C H,GAO,et al.Novel roles of Skp2 E3 ligase in cellular senescence,cancer progression,and metastasis[J].Chin J Cancer,2012,31(4):169-177.DOI:10.5732/cjc.011.10319.
[9]WEI Z,JIANG X,LIU F,et.al.Downregulation of SKP2 inhibits the growth and metastasis of gastric cancer cells in vitro and in vivo[J].Tumour Biol,2013,34(1):181-192.DOI:10.1007/s13277-012-0527-8.
[10]MORROW J K,LIN H K,SUN S C,et al.Targeting ubiquitination for cancer therapies[J].Future Med Chem,2015,7(17):2333-2350.DOI:10.4155/fmc.15.148.
[11]OH M,LEE J H,MOON H,et al.A chemical inhibitor of the Skp2/p300 interaction that promotes p53-mediated apoptosis[J].Angewandte Chemie,2016,55(2):602-606.DOI:10.1002/anie.201508716.
[12]CHAN C H,MORROW J K,LI C F,et al.Pharmacological inactivation of SKP2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression[J].Cell,2013,154(3):556-558.DOI:10.1016/j.cell.2013.06.048.
[13]UNGERMANNOVA D,LEE J,ZHANG G,et al.High-throughput screening alpha screen assay for identification of small-molecule inhibitors of ubiquitin E3 ligase SCFSkp2-Cks1[J].J Biomolecular Screening,2013,18(8):910-920.DOI:10.1177/1087057 113485789.
[14]CHAN C H,LI C F,YANG W L,et al.The Skp2-SCF E3 ligase regulates Akt ubiquitination,glycolysis,herceptin sensitivity,and tumorigenesis[J].Cell,2012,149(5):1098-1111.DOI:10.1016/j.cell.2012.10.025.
[15]LU H,CAO X,ZHANG H,et al.Imbalance between MMP-2,9and TIMP-1 promote the invasion and metastasis of renal cell carcinoma via SKP2 signaling pathways[J].Tumour Biol,2014,35(10):9807-9813.DOI:10.1007/s13277-014-2256-7.
[16]BHATTACHARYA S,GARRIGA J,CALBO J,et al.SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells[J].Oncogene,2003,22(16):2443-2451.DOI:10.1038/sj.onc.1206339.
[17]HUANG H,SONG Y,WU Y,et al.Erbin loss promotes cancer cell proliferation through feedback activation of Akt-Skp2-p27 signaling[J].Biochem Biophys Res Commun,2015,463(3):370-376.DOI:10.1016/j.bbrc.2015.05.071.