HDAC2 sumo-E3连接酶活性在DLD1细胞迁移和增殖中的作用
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摘要
目的:探讨HDAC2 sumo-E3连接酶对DLD1细胞迁移与增殖的影响和可能机制。方法:全基因合成HDAC2 sumo-E3连接酶突变体HDAC2(325-488),并构建其重组慢病毒表达载体;感染DLD1hdac2-/-细胞(无内源性HDAC2表达),筛选稳定表HDAC2(325-488)的细胞克隆DLD1h325-488;RTCA实时细胞分析仪、Trans-well等检测DLD1h325-488稳定细胞的增殖和迁移能力;免疫印迹、q RT-PCR检测DLD1h325-488稳定细胞增殖和迁移相关基因的表达。结果:构建了稳定表达HDAC2 sumo-E3连接酶突变体HDAC2(325-488)的细胞株DLD1h325-488,并获得稳定表达HDAC2 sumo-E3连接酶突变体HDAC2(325-488)的DLD1h325-488细胞。与对照组细胞相比,DLD1h325-488细胞增殖活性无显著变化,但是细胞的迁移能力和迁移相关蛋白MMP14的表达显著上升。结论:HDAC2 sumo-E3连接酶可能通过上调mmp14的表达而促进DLD1细胞的迁移。
Objective: To investigate the effect of HDAC2 sumo-E3 ligase segment on proliferation and migration of DLD1 cells and the potential mechanisms.Methods: The coding sequence of the sumo-E3 ligase segment of HDAC2(325-488 aa) was chemically synthesized and ligated into a recombinant lentiviral vector.The lentiviral vector was packaged into lentiviral particles and used to infect HDAC2-knockout DLD1 cells(DLD1-HDAC2-/-),and stably transfected cells were selected and named DLD1 h325-488.Then,proliferation and migration of the DLD1 h325-488 cells were determined by RTCA and transwell assay; and changes in expression of proliferation-and migration-related genes were determined by q RT-PCR and immune-blotting.Results: Stable DLD1 h325-488 cell line was successfully obtained.Compared to the control cells,and DLD1 h325-488 cells stably expressing HDAC2 sumo-E3 ligase mutant.HDAC2(325-488).DLD1 h325-488 cells showed no significant changes in proliferation,but significantly increased migration,which was accompanied by increase expression of the migration-related MMP14.Conclusion: HDAC2 sumo-E3 ligase segment may promote the migration of DLD1 cells by upregulating MMP14 expression.
Objective: To investigate the effect of HDAC2 sumo-E3 ligase segment on proliferation and migration of DLD1 cells and the potential mechanisms.Methods: The coding sequence of the sumo-E3 ligase segment of HDAC2(325-488 aa) was chemically synthesized and ligated into a recombinant lentiviral vector.The lentiviral vector was packaged into lentiviral particles and used to infect HDAC2-knockout DLD1 cells(DLD1-HDAC2-/-),and stably transfected cells were selected and named DLD1 h325-488.Then,proliferation and migration of the DLD1 h325-488 cells were determined by RTCA and transwell assay; and changes in expression of proliferation-and migration-related genes were determined by q RT-PCR and immune-blotting.Results: Stable DLD1 h325-488 cell line was successfully obtained.Compared to the control cells,and DLD1 h325-488 cells stably expressing HDAC2 sumo-E3 ligase mutant.HDAC2(325-488).DLD1 h325-488 cells showed no significant changes in proliferation,but significantly increased migration,which was accompanied by increase expression of the migration-related MMP14.Conclusion: HDAC2 sumo-E3 ligase segment may promote the migration of DLD1 cells by upregulating MMP14 expression.
引文
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[28]Yi SA,Ryu HW,Lee DH,et al.HP1beta suppresses metastasis of human cancer cells by decreasing the expression and activation of MMP2[J].Int J Oncol,2014,45(6):41-48
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[32]Lin KT,Wang YW,Chen CT,et al.HDAC inhibitors augmented cell migration and metastasis through induction of PKCs leading to identification of low toxicity modalities for combination cancer therapy[J]Clin Cancer Res,2012,18(17):691-701
[33]Ji M,Lee EJ,Kim KB,et al.HDAC inhibitors induce epithelial-mesenchymal transition in colon carcinoma cells[J].Oncol Rep,2015,33(5):299-308
[2]Schmitz ML,de la Vega L.New Insights into the Role of Histone Deacetylases as Coactivators of Inflammatory Gene Expression[J].Antioxid Redox Signal,2015,23(1):85-98
[3]Morris MJ,Monteggia LM.Unique functional roles for class I and class II histone deacetylases in central nervous system development and function[J].Dev Neurosci,2013,31(6):70-81
[4]Kee HJ,Kook H.Roles and targets of class I and IIa histone deacetylases in cardiac hypertrophy[J].Biomed Biotechnol,2011,2011:928326
[5]Haberland M,Montgomery RL,Olson EN.The many roles of histone deacetylases in development and physiology:implications for disease and therapy[J].Nat Rev Genet,2009,10(1):32-42
[6]Segre CV,Chiocca S.Regulating the regulators:the post-translational code of class I HDAC1 and HDAC2[J].Biomed Biotechnol,2011,2011:690848
[7]Shan W,Jiang Y,Yu H,et al.HDAC2 overexpression correlates with aggressive clinicopathological features and DNA-dam age response pathway of breast cancer[J].Am J Cancer Res,2017,7(5):1213-1226
[8]Kobayashi T,Nakazono K,Tokuda M,et al.HDAC2 promotes loss of primary cilia in pancreatic ductal adenocarcinoma[J].EMBO Rep,2017,18(2):334-343
[9]Muller BM,Jana L,Kasajima A,et al.Differential expression of histone deacetylases HDAC1,2 and 3 in human breast cancer--overexpression of HDAC2 and HDAC3 is associated with clinicopathological indicators of disease progression[J].BMC Cancer,2013,13:215
[10]Ashktorab H,Belgrave K,Hosseinkhah F,et al.Global histone H4acetylation and HDAC2 expression in colon adenoma and carcinoma[J].Dig Dis Sci,2009,54(10):09-17
[11]Haery L,Thompson RC,Gilmore TD.Histone acetyltransferases and histone deacetylases in B-and T-cell development,physiology and malignancy[J].Genes Cancer,2015,6(5-6):184-213
[12]Li Z,Hao Q,Luo J,et al.USP4 inhibits p53 and NF-kappa B through deubiquitinating and stabilizing HDAC2[J].Oncogene,2016,35(22):02-12
[13]Brandl A,Wagner T,Uhilq KM,et al.Dynamically regulated sumoylation of HDAC2 controls p53 deacetylation and restricts apoptosis following genotoxic stress[J].Mol Cell Biol,2012,4(5):84-93
[14]Kim JK,Noh JH,Eun JW,et al.Targeted inactivation of HDAC2 restores p16INK4a activity and exerts antitumor effects on human gastric cancer[J].Mol Cancer Res,2013,11(1):62-73
[15]Bianco S,Jangal M,Garneau D,et al.LRH-1 controls proliferation in breast tumor cells by regulating CDKN1A gene expression[J].Oncogene,2015,34(34):09-18
[16]Pluemsampant S,Safronova OS,Nakahama K,et al.Protein kinase CK2 is a key activator of histone deacetylase in hypoxia-associated tumors[J].Int J Cancer,2008,122(2):33-41
[17]Ropero S,Ballestar E,Alaminos M,et al.Transforming pathways unleashed by a HDAC2 mutation in human cancer[J].Oncogene,2008,27(28):08-12
[18]Zhu P,Martin E,Mengwasser J,et al.Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis[J].Cancer Cell,2004,5(5):55-63
[19]Zimmermann S,Kiefer F,Prudenziati M,et al.Reduced body size and decreased intestinal tumor rates in HDAC2-mutant mice[J].Cancer Res,2007,67(19):47-54
[20]Xu X,Vatsvavan J,Gao C,et al.HDAC2 promotes e IF4E sumoylation and activates m RNA translation gene specifically[J].Biol Chem,2010,285(24):39-43
[21]Xu X,Vatsvavan J,Gao C,et al.Sumoylation of e IF4E activates m RNA translation[J].EMBO Rep,2010,11(4):299-304
[22]Chen LZ,Li XY,Huang H,et al.SUMO-2 promotes m RNA translation by enhancing interaction between e IF4E and e IF4G[J].PLo S One,2014,9(6):04-57
[23]郭韡,熊渊,黄宏,等.组蛋白去乙酰化酶HDAC2突变体构建及其SUMO修饰E3连接酶功能研究[J].现代生物医学进展,2013,13(6):5-8Guo Wei,Xiong Yuan,Huang Hong,et al.Construction of histone deacetylase HDAC2 mutant and its function of SUMO-modified E3ligase[J].Progress in Modern Biomedicine,2013,13(6):5-8
[24]Liu Y,Li S,Zhang H,et al.A one-step cloning method for the construction of somatic cell gene targeting vectors:application to production of human knockout cell lines[J].BMC Biotechnol,2012,12:71
[25]Chen W,Xia T,Wang D,et al.Human astrocytes secrete IL-6 to promote glioma migration and invasion through upregulation of cytomembrane MMP14[J].Oncotarget,2016,7(38):62425-62438
[26]Hui P,Xu X,Xu L,et al.Expression of MMP14 in invasive pituitary adenomas:relationship to invasion and angiogenesis[J].Int J Clin Exp Pathol,2015,8(4):56-67
[27]Lu H,Hu L,Yu L,et al.KLF8 and FAK cooperatively enrich the active MMP14 on the cell surface required for the metastatic progression of breast cancer[J].Oncogene,2014,33(22):09-17
[28]Yi SA,Ryu HW,Lee DH,et al.HP1beta suppresses metastasis of human cancer cells by decreasing the expression and activation of MMP2[J].Int J Oncol,2014,45(6):41-48
[29]Sakamoto T,Weng JS,Hara T,et al.Hypoxia-inducible factor 1 regulation through cross talk between m TOR and MT1-MMP[J].Mol Cell Biol,2014,34(1):30-42
[30]West AC,Johnstone RW.New and emerging HDAC inhibitors for cancer treatment[J].Clin Invest,2014,124(1):0-9
[31]Spiegel S,Milstien S,Grant S.Endogenous modulators and pharmacological inhibitors of histone deacetylases in cancer thera py[J].Oncogene,2012,31(5):37-51
[32]Lin KT,Wang YW,Chen CT,et al.HDAC inhibitors augmented cell migration and metastasis through induction of PKCs leading to identification of low toxicity modalities for combination cancer therapy[J]Clin Cancer Res,2012,18(17):691-701
[33]Ji M,Lee EJ,Kim KB,et al.HDAC inhibitors induce epithelial-mesenchymal transition in colon carcinoma cells[J].Oncol Rep,2015,33(5):299-308