泛素蛋白连接酶E3A在乳腺癌细胞中作用的蛋白质组学与生物信息学分析
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摘要
目的:通过蛋白质组学与生物信息学方法探讨泛素蛋白连接酶E3A(UBE3A)在乳腺癌细胞中的生物学功能。方法:将三阴性乳腺癌细胞MDA-MB-231分别转染shRNA-UBE3A片段(UBE3A敲低组)与阴性对照shRNA片段(对照组)后,采用双向电泳(2-DE)法及基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-TOF-MS/MS)对两组细胞的差异蛋白质分离和鉴定;采用DAVID Functional Annotation及String在线工具对差异表达蛋白质进行生物信息学分析。结果:2-DE及MALDI-TOF-TOF-MS/MS分离和鉴定出28个差异蛋白,其中UBE3A敲低组相对于对照组有4个表达上调,24个表达下调。与DAVID数据库有23个相匹配,其中能够进行生物学途径、细胞成分、分子功能注释分类的蛋白质分别为23个(100%)、20个(87.7%)和23个(100%),而在泛素-蛋白酶体系统及糖代谢方面分类比较明确;String分析显示这些差异蛋白中有23个存在直接或间接的联系。结论:UBE3A可能通过泛素-蛋白酶体系统功能与糖代谢途径参与乳腺癌细胞生物学行为的调控。
Objective: To investigate the potential biological function of ubiquitin-protein ligase E3A(UBE3A) in breast cancer cells through proteomics and bioinformatics methods. Methods: The triple-negative breast cancer MDA-MB-231 cells were transfected with shRNA-UBE3A sequences(UBE3A knock-down group) or scrambled shRNA sequences(control group), respectively. Then, the differentially expressed proteins between the two groups of cells were separated and identified by twodimensional electrophoresis(2-DE) and matrix-assisted laser desorption/ionization(MALDI-TOF-TOF-MS/MS); bioinformatics analyses of the differentially expressed proteins were performed using DAVID Functional Annotation and String online tools.Results: Twenty-eight differentially expressed proteins were obtained and identified by 2-DE and MALDI-TOFTOF-MS/MS, and 23 proteins were matched in DAVID database, in which, 4 were up-regulated, 24 were downregulated in UBE3A knock-down group versus control group. Twenty-three proteins were recognized by DAVID database, in which, 23(100%), 20(87.7%) and 23(100%) proteins could be annotated by biological pathway, cellular components and annotation of molecule functions especially, some of them were clearly related to the ubiquitin-proteasome system and glucose metabolism. String analysis showed that there were direct or indirect relations among the 23 proteins.Conclusion: UBE3A may participate in regulation of the biological behaviors of breast cancer cells via the ubiquitin-proteasome system and glucose metabolism pathway.
Objective: To investigate the potential biological function of ubiquitin-protein ligase E3A(UBE3A) in breast cancer cells through proteomics and bioinformatics methods. Methods: The triple-negative breast cancer MDA-MB-231 cells were transfected with shRNA-UBE3A sequences(UBE3A knock-down group) or scrambled shRNA sequences(control group), respectively. Then, the differentially expressed proteins between the two groups of cells were separated and identified by twodimensional electrophoresis(2-DE) and matrix-assisted laser desorption/ionization(MALDI-TOF-TOF-MS/MS); bioinformatics analyses of the differentially expressed proteins were performed using DAVID Functional Annotation and String online tools.Results: Twenty-eight differentially expressed proteins were obtained and identified by 2-DE and MALDI-TOFTOF-MS/MS, and 23 proteins were matched in DAVID database, in which, 4 were up-regulated, 24 were downregulated in UBE3A knock-down group versus control group. Twenty-three proteins were recognized by DAVID database, in which, 23(100%), 20(87.7%) and 23(100%) proteins could be annotated by biological pathway, cellular components and annotation of molecule functions especially, some of them were clearly related to the ubiquitin-proteasome system and glucose metabolism. String analysis showed that there were direct or indirect relations among the 23 proteins.Conclusion: UBE3A may participate in regulation of the biological behaviors of breast cancer cells via the ubiquitin-proteasome system and glucose metabolism pathway.
引文
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[5]Zhou X,Deng S,Liu H,et al.Knockdown of ubiquitin protein ligase E3A affects proliferation and invasion,and induces apoptosis of breast cancer cells through regulation of annexin A2[J].Mol Med Rep,2015,12(1):1107-1113.doi:10.3892/mmr.2015.3549.
[6]Krishnan V,Stoppel DC,Nong Y,et al.Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1[J].Nature,2017,543(7646):507-512.doi:10.1038/nature21678.
[7]Lopez SJ,Segal DJ,LaSalle JM.UBE3A:An E3 Ubiquitin Ligase With Genome-Wide Impact in Neurodevelopmental Disease[J].Front Mol Neurosci,2019,11:476.doi:10.3389/fnmol.2018.00476.
[8]Carpentier SC.Multiple Testing and Pattern Recognition in 2-DEProteomics[J].Methods Mol Biol,2016,1384:215-235.doi:10.1007/978-1-4939-3255-9_13.
[9]Westermeier R,G?rg A.Two-dimensional electrophoresis in proteomics[J].Methods Biochem Anal,2011,54:411-439.
[10]Szklarczyk D,Morris JH,Cook H,et al.The STRING database in2017:quality-controlled protein-protein association networks,made broadly accessible[J].Nucleic Acids Res,2017,45(D1):D362-368.doi:10.1093/nar/gkw937.
[11]Giovannucci E,Harlan DM,Archer MC,et al.Diabetes and cancer:a consensus report[J].CA Cancer J Clin,2010,60(4):207-221.doi:10.3322/caac.20078.
[12]Kyu HH,Bachman VF,Alexander LT,et al.Physical activity and risk of breast cancer,colon cancer,diabetes,ischemic heart disease,and ischemic stroke events:systematic review and dose-response meta-analysis for the Global Burden of Disease Study 2013[J].BMJ,2016,354:i3857.doi:10.1136/bmj.i3857.
[13]Gallagher EJ,LeRoith D.Obesity and Diabetes:The Increased Risk of Cancer and Cancer-Related Mortality[J].Physiol Rev,2015,95(3):727-748.doi:10.1152/physrev.00030.2014.
[14]Samuel SM,Varghese E,Varghese S,et al.Challenges and perspectives in the treatment of diabetes associated breast cancer[J].Cancer Treat Rev,2018,70:98-111.doi:10.1016/j.ctrv.2018.08.004.
[15]Hatoum D,McGowan EM.Recent advances in the use of metformin:can treating diabetes prevent breast cancer?[J].Biomed Res Int,2015,2015:548436.doi:10.1155/2015/548436.
[16]De Bruijn KM,Arends LR,Hansen BE,et al.Systematic review and meta-analysis of the association between diabetes mellitus and incidence and mortality in breast and colorectal cancer[J].Br JSurg,2013,100(11):1421-1429.doi:10.1002/bjs.9229.
[17]Prasanth KR,Chuang C,Nagy PD.Co-opting ATP-generating glycolytic enzyme PGK1 phosphoglycerate kinase facilitates the assembly of viral replicase complexes[J].PLoS Pathog,2017,13(10):e1006689.doi:10.1371/journal.ppat.1006689.
[18]Yuan SX,Yang F,Yang Y,et al.Long noncoding RNA associated with microvascular invasion in hepatocellular carcinoma promotes angiogenesis and serves as a predictor for hepatocellular carcinoma patients'poor recurrence-free survival after hepatectomy[J].Hepatology,2012,56(6):2231-2241.doi:10.1002/hep.25895.
[19]Dou C,Zheng X,Liu Q,et al.Is the regulatory effect of PCAFand sirtuin 7 on phosphoglycerate kinase 1 acetylation a universal mechanism underlying hepatocellular carcinoma progression?[J].Hepatology,2017,66(5):1699-1700.doi:10.1002/hep.29351.
[20]Ahmad SS,Glatzle J,Bajaeifer K,et al.Phosphoglycerate kinase1 as a promoter of metastasis in colon cancer[J].Int J Oncol,2013,43(2):586-90.doi:10.3892/ijo.2013.1971.
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