遗传 ›› 2016, Vol. 38 ›› Issue (2): 93-102.doi: 10.16288/j.yczz.15-470
• 综述 • 下一篇
刘茜1,王瑾晖2,李晓宇1,岑山1
收稿日期:2015-11-16
修回日期:2015-12-02
出版日期:2016-02-20
发布日期:2016-01-05
通讯作者:
李晓宇,博士,副研究员,研究方向：病毒学。E-mail: xiaoyulik@hotmail.com作者简介:刘茜,硕士研究生,专业方向：MOV10抑制LINE-1分子机制的研究。E-mail: shine_lqian@163.com
基金资助:Qian Liu1, Jinhui Wang2, Xiaoyu Li1, Shan Cen1
Received:2015-11-16
Revised:2015-12-02
Online:2016-02-20
Published:2016-01-05
摘要： LINE-1是现今人体内存在的唯一具有自主转座活性的转座子,约有500 000个拷贝,占人类基因组总量的17%。LINE-1是通过转录和逆转录在内的转座过程产生新的DNA拷贝,并使新产生的DNA拷贝插入基因组的不同位置。LINE-1转座会影响基因组中其他基因的表达或调控,因而会对基因组的稳定性产生影响,从而导致基因疾病或肿瘤的发生。本文总结了近年来国际上对LINE-1转座与肿瘤的发生和发展之间关系的研究进展,为肿瘤的治疗和机制研究提供一些线索。
刘茜,王瑾晖,李晓宇,岑山. 逆转录转座子LINE-1与肿瘤的发生和发展[J]. 遗传, 2016, 38(2): 93-102.
Qian Liu, Jinhui Wang, Xiaoyu Li, Shan Cen. The connection between LINE-1 retrotransposition and human tumorigenesis[J]. HEREDITAS(Beijing), 2016, 38(2): 93-102.
| [1] Unwin N. The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission. Philos Trans R Soc Lond B Biol Sci , 2000, 355(1404): 1813-1829. [2] McClintock B. The origin and behavior of mutable loci in maize. Proc Natl Acad Sci USA , 1950, 36(6): 344-355. [3] Goodier JL, Kazazian HH Jr. Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell , 2008, 135(1): 23-35. [4] Bao WD, Kojima KK, Kohany O. Repbase Update, a database of repetitive elements in eukaryotic genomes. Mob DNA , 2015, 6: 11. [5] Blikstad V, Benachenhou F, Sperber GO, Blomberg J. Endogenous retroviruses-evolution of human endogenous retroviral sequences: a conceptual account. Cell Mol Life Sci , 2008, 65(21): 3348-3365. [6] Belancio VP, Hedges DJ, Deininger P. Mammalian non-LTR retrotransposons: for better or worse, in sickness and in health. Genome Res , 2008, 18(3): 343-358. [7] Beck CR, Garcia-Perez JL, Badge RM, Moran JV. LINE-1 elements in structural variation and disease. Annu Rev Genomics Hum Genet , 2011, 12: 187-215. [8] Swergold GD. Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol Cell Biol , 1990, 10(12): 6718-6729. [9] Severynse DM, Hutchison CA 3rd, Edgell MH. Identification of transcriptional regulatory activity within the 5' A-type monomer sequence of the mouse LINE-1 retroposon. Mamm Genome , 1992, 2(1): 41-50. [10] Feng QH, Moran JV, Kazazian HH Jr, Boeke JD. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell , 1996, 87(5): 905-916. [11] Martin SL, Li JF, Epperson LE, Lieberman B. Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis. Gene , 1998, 215(1): 69-75. [12] Moran JV, Holmes SE, Naas TP, DeBerardinis RJ, Boeke JD, Kazazian HH Jr. High frequency retrotransposition in cultured mammalian cells. Cell , 1996, 87(5): 917-927. [13] Belgnaoui SM, Gosden RG, Semmes OJ, Haoudi A. Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells. Cancer Cell Int , 2006, 6: 13. [14] Babushok DV, Kazazian HH Jr. Progress in understanding the biology of the human mutagen LINE-1. Hum Mutat , 2007, 28(6): 527-539. [15] Farkash EA, Kao GD, Horman SR, Luning Prak ET. Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay. Nucleic Acids Res , 2006, 34(4): 1196-1204. [16] Gasior SL, Wakeman TP, Xu B, Deininger PL. The human LINE-1 retrotransposon creates DNA double-strand breaks. J Mol Bio , 2006, 357(5): 1383-1393. [17] Denli AM, Narvaiza I, Kerman BE, Pena M, Benner C, Marchetto MCN, Diedrich JK, Aslanian A, Ma J, Moresco JJ, Moore L, Hunter T, Saghatelian A, Gage FH. Primate-specific ORF0 contributes to retrotransposon-mediated diversity. Cell , 2015, 163(3): 583-593. [18] Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet , 1997, 13(8): 335-340. [19] Bourc'his D, Bestor TH. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature , 2004, 431(7004):96-99. [20] Kato Y, Kaneda M, Hata K, Kumaki K, Hisano M, Kohara Y, Okano M, Li E, Nozaki M, Sasaki H. Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse. Hum Mol Genet , 2007, 16(19): 2272-2280. [21] Branciforte D, Martin SL. Developmental and cell type specificity of LINE-1 expression in mouse testis: implications for transposition. Mol Cell Biol , 1994, 14(4): 2584-2592. [22] Kano H, Godoy I, Courtney C, Vetter MR, Gerton GL, Ostertag EM, Kazazian HH Jr. L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism. Genes Dev , 2009, 23(11): 1303-1312. [23] Garcia-Perez JL, Marchetto MCN, Muotri AR, Coufal NG, Gage FH, O'Shea KS, Moran JV. LINE-1 retrotransposition in human embryonic stem cells. Hum Mol Genet , 2007, 16(13): 1569-1577. [24] van den Hurk JAJM, Meij IC, Seleme MC, Kano H, Nikopoulos K, Hoefsloot LH, Sistermans EA, de Wijs IJ, Mukhopadhyay A, Plomp AS, de Jong PTVM, Kazazian HH, Cremers FPM. L1 retrotransposition can occur early in human embryonic development. Hum Mol Genet , 2007, 16(13): 1587-1592. [25] Chiu YL, Greene WC. The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu Rev Immunol , 2008, 26: 317-353. [26] Narvaiza I, Linfesty DC, Greener BN, Hakata Y, Pintel DJ, Logue E, Landau NR, Weitzman MD. Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase. PLoS Pathog , 2009, 5(5): e1000439. [27] Renard M, Henry M, Guétard D, Vartanian JP, Wain- Hobson S. APOBEC1 and APOBEC3 cytidine deaminases as restriction factors for hepadnaviral genomes in non- humans in vivo. J Mol Biol , 2010, 400(3): 323-334. [28] Bogerd HP, Wiegand HL, Hulme AE, Garcia-Perez JL, Sue O'Shea K, Moran JV, Cullen BR. Cellular inhibitors of long interspersed element 1 and Alu retrotransposition. Proc Natl Acad Sci USA , 2006, 103(23): 8780-8785. [29] Esnault C, Millet J, Schwartz O, Heidmann T. Dual inhibitory effects of APOBEC family proteins on retrotransposition of mammalian endogenous retroviruses. Nucleic Acids Res , 2006, 34(5): 1522-1531. [30] Chen H, Lilley CE, Yu Q, Lee DV, Chou J, Narvaiza I, Landau NR, Weitzman MD. APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons. Curr Biol , 2006, 16(5): 480-485. [31] Li XY, Zhang JY, Jia R, Cheng V, Xu X, Qiao WT, Guo F, Liang C, Cen S. The MOV10 helicase inhibits LINE-1 mobility. J Biol Chem , 2013, 288(29): 21148-211460. [32] Arjan-Odedra S, Swanson CM, Sherer NM, Wolinsky SM, Malim MH. Endogenous MOV10 inhibits the retrotransposition of endogenous retroelements but not the replication of exogenous retroviruses. Retrovirology , 2012, 9: 53. [33] Goodier JL, Cheung LE, Kazazian HH Jr. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells. PLoS Genet , 2012, 8(10): e1002941. [34] Hu SQ, Li J, Xu FW, Mei S, Le Duff Y, Yin LJ, Pang XJ, Cen S, Jin Q, Liang C, Guo F. SAMHD1 Inhibits line-1 retrotransposition by promoting stress granule formation. PLoS Genet , 2015, 11(7): e1005367. [35] Zhao K, Du J, Han X, Goodier JL, Li P, Zhou XH, Wei W, Evans SL, Li LZ, Zhang WY, Cheung LE, Wang GJ, Kazazian HH Jr, Yu XF. Modulation of LINE-1 and Alu/SVA retrotransposition by Aicardi-Goutières syndrome- related SAMHD1. Cell Rep , 2013, 4(6): 1108-1115. [36] Goodier JL, Pereira GC, Cheung LE, Rose RJ, Kazazian HH Jr. The broad-spectrum antiviral protein ZAP restricts human retrotransposition. PLoS Genet , 2015, 11(5): e1005252. [37] Van Meter M, Kashyap M, Rezazadeh S, Geneva AJ, Morello TD, Seluanov A, Gorbunova V. SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age. Nat Commun , 2014, 5: 5011. [38] Roman AC, Benitez DA, Carvajal-Gonzalez JM, Fernandez-Salguero PM. Genome-wide B1 retrotransposon binds the transcription factors dioxin receptor and Slug and regulates gene expression in vivo . Proc Natl Acad Sci USA , 2008, 105(5): 1632-1637. [39] Gilbert N, Lutz-Prigge S, Moran JV. Genomic deletions created upon LINE-1 retrotransposition. Cell , 2002, 110(3): 315-325. [40] Symer DE, Connelly C, Szak ST, Caputo EM, Cost GJ, Parmigiani G, Boeke JD. Human l1 retrotransposition is associated with genetic instability in vivo . Cell , 2002, 110(3): 327-338. [41] Miné M, Chen JM, Brivet M, Desguerre I, Marchant D, de Lonlay P, Bernard A, Férec C, Abitbol M, Ricquier D, Marsac C. A large genomic deletion in the PDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element. Hum Mutat , 2007, 28(2): 137-142. [42] Takasu M, Hayashi R, Maruya E, Ota M, Imura K, Kougo K, Kobayashi C, Saji H, Ishikawa Y, Asai T, Tokunaga K. Deletion of entire HLA-A gene accompanied by an insertion of a retrotransposon. Tissue Antigens , 2007, 70(2): 144-150. [43] Kazazian HH Jr., Wong C, Youssoufian H, Scott AF, Phillips DG, Antonarakis SE. Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature , 1988, 332(6160): 164-166. [44] Morse B, Rotherg PG, South VJ, Spandorfer JM, Astrin SM. Insertional mutagenesis of the myc locus by a LINE-1 sequence in a human breast carcinoma. Nature , 1988, 333(6168): 87-90. [45] Miki Y, Nishisho I, Horii A, Miyoshi Y, Utsunomiya J, Kinzler KW, Vogelstein B, Nakamura Y. Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res , 1992, 52(3): 643-645. [46] Solyom S, Ewing AD, Rahrmann EP, Doucet T, Nelson HH, Burns MB, Harris RS, Sigmon DF, Casella A, Erlanger B, Wheelan S, Upton KR, Shukla R, Faulkner GJ, Largaespada DA, Kazazian HH Jr. Extensive somatic L1 retrotransposition in colorectal tumors. Genome Res , 2012, 22(12): 2328-2338. [47] Lee E, Iskow R, Yang LX, Gokcumen O, Haseley P, Luquette LJ III, Lohr JG, Harris CC, Ding L, Wilson RK, Wheeler DA, Gibbs RA, Kucherlapati R, Lee C, Kharchenko PV, Park PJ. Landscape of somatic retrotransposition in human cancers. Science , 2012, 337(6097): 967- 971. [48] Iskow RC, McCabe MT, Mills RE, Torene S, Stephen Pittard W, Neuwald AF, Van Meir EG, Vertino PM, Devine SE. Natural mutagenesis of human genomes by endogenous retrotransposons. Cell , 2010, 141(7): 1253-1261. [49] Shukla R, Upton KR, Muñoz-Lopez M, Gerhardt DJ, Fisher ME, Nguyen T, Brennan PM, Kenneth Baillie J, Collino A, Ghisletti S, Sinha S, Iannelli F, Radaelli E, Dos Santos A, Rapoud D, Guettier C, Samuel D, Natoli G, Carninci P, Ciccarelli FD, Garcia-Perez JL, Faivre J, Faulkner GJ. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell , 2013, 153(1): 101-111. [50] Rodić N, Sharma R, Sharma R, Zampella J, Dai LX, Taylor MS, Hruban RH, Iacobuzio-Donahue CA, Maitra A, Torbenson MS, Goggins M, Shih IM, Duffield AS, Montgomery EA, Gabrielson E, Netto GJ, Lotan TL, De Marzo AM, Westra W, Binder ZA, Orr BA, Gallia GL, Eberhart CG, Boeke JD, Harris CR, Burns KH. Long interspersed element-1 protein expression is a hallmark of many human cancers. Am J Pathol , 2014, 184(5): 1280- 1286. [51] Wallace NA, Belancio VP, Deininger PL. L1 mobile element expression causes multiple types of toxicity. Gene , 2008, 419(1-2): 75-81. [52] Lau CC, Sun TT, Ching AKK, He M, Li JW, Wong AM, Co NN, Chan AWH, Li PS, Lung RWM, Tong JHM, Lai PBS, Chan HLY, To KF, Chan TF, Wong N. Viral- human chimeric transcript predisposes risk to liver cancer development and progression. Cancer cell , 2014, 25(3): 335-349. [53] Bréchot C, Gozuacik D, Murakami Y, Paterlini-Bréchot P. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin Cancer Biol , 2000, 10(3): 211-231. [54] Ding D, Lou XY, Hua DS, Yu W, Li LS, Wang J, Gao F, Zhao N, Ren GP, Li LJ, Lin BY. Recurrent targeted genes of hepatitis B virus in the liver cancer genomes identified by a next-generation sequencing-based approach. PLoS Genet , 2012, 8(12): e1003065. [55] Harris CR, Normart R, Yang QF, Stevenson E, Haffty BG, Ganesan S, Cordon-Cardo C, Levine AJ, Tang LH. Association of nuclear localization of a long interspersed nuclear element-1 protein in breast tumors with poor prognostic outcomes. Genes Cancer , 2010, 1(2): 115-124. [56] Su YH, Davies S, Davis M, Lu H, Giller R, Krailo M, Cai QY, Robison L, Shu XO. Children's Oncology Group. Expression of LINE-1 p40 protein in pediatric malignant germ cell tumors and its association with clinicopathological parameters: a report from the Children's Oncology Group. Cancer letter , 2007, 247(2): 204-212. [57] Belancio VP, Roy-Engel AM, Pochampally RR, Deininger P. Somatic expression of LINE-1 elements in human tissues. Nucleic Acids Res , 2010, 38(12): 3909-3922. [58] Ting DT, Lipson D, Paul S, Brannigan BW, Akhavanfard S, Coffman EJ, Contino G, Deshpande V, Iafrate AJ, Letovsky S, Rivera MN, Bardeesy N, Maheswaran S, Haber DA. Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers. Science , 2011, 331(6017): 593-596. [59] Matsunoki A, Kawakami K, Kotake M, Kaneko M, Kitamura H, Ooi A, Watanabe G, Minamoto T. LINE-1 methylation shows little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer. BMC Cancer , 2012, 12: 574. [60] Sciamanna I, Vitullo P, Curatolo A, Spadafora C. A reverse transcriptase-dependent mechanism is essential for murine preimplantation development. Genes , 2011, 2(2): 360-373. [61] Mangiacasale R, Pittoggi C, Sciamanna I, Careddu A, Mattei E, Lorenzini R, Travaglini L, Landriscina M, Barone C, Nervi C, Lavia P, Spadafora C. Exposure of normal and transformed cells to nevirapine, a reverse transcriptase inhibitor, reduces cell growth and promotes differentiation. Oncogene , 2003, 22(18): 2750-2761. [62] Landriscina M, Fabiano A, Altamura S, Bagalà C, Piscazzi A, Cassano A, Spadafora C, Giorgino F, Barone C, Cignarelli M. Reverse transcriptase inhibitors down-regulate cell proliferation in vitro and in vivo and restore thyrotropin signaling and iodine uptake in human thyroid anaplastic carcinoma. J Clin Endocrinol Metab , 2005, 90(10): 5663-5671. [63] Sciamanna I, Landriscina M, Pittoggi C, Quirino M, Mearelli C, Beraldi R, Mattei E, Serafino A, Cassano A, Sinibaldi-Vallebona P, Garaci E, Barone C, Spadafora C. Inhibition of endogenous reverse transcriptase antagonizes human tumor growth. Oncogene , 2005, 24(24): 3923-3931. [64] Oricchio E, Sciamanna I, Beraldi R, Tolstonog GV, Schumann GG, Spadafora C. Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression. Oncogene , 2007, 26(29): 4226-4233. [65] Sciamanna I, Gualtieri A, Cossetti C, Osimo EF, Ferracin M, Macchia G, Aricò E, Prosseda G, Vitullo P, Misteli T, Spadafora C. A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines. Oncotarget , 2013, 4(12): 2271-2287. [66] Weber B, Kimhi S, Howard G, Eden A, Lyko F. Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription. Oncogene , 2010, 29(43): 5775-5784. [67] Pattamadilok J, Huapai N, Rattanatanyong P, Vasurattana A, Triratanachat S, Tresukosol D, Mutirangura A. LINE-1 hypomethylation level as a potential prognostic factor for epithelial ovarian cancer. Int J Gynecol Cancer , 2008, 18(4): 711-717. [68] Sunami E, de Maat M, Vu A, Turner RR, Hoon DSB. LINE-1 hypomethylation during primary colon cancer progression. PloS One , 2011, 6(4): e18884. [69] Pavicic W, Joensuu EI, Nieminen T, Peltomäki P. LINE-1 hypomethylation in familial and sporadic cancer. J Mol Med (Berl) , 2012, 90(7): 827-835. [70] Antelo M, Balaguer F, Shia J, Shen Y, Hur K, Moreira L, Cuatrecasas M, Bujanda L, Giraldez MD, Takahashi M, Cabanne A, Barugel ME, Arnold M, Roca EL, Andreu M, Castellvi-Bel S, Llor X, Jover R, Castells A, Boland CR, Goel A. A high degree of LINE-1 hypomethylation is a unique feature of early-onset colorectal cancer. PLoS One , 2012, 7(9): e45357. [71] Gao Y, Baccarelli A, Shu XO, Ji BT, Yu K, Tarantini L, Yang G, Li HL, Hou L, Rothman N, Zheng W, Gao YT, Chow WH. Blood leukocyte Alu and LINE-1 methylation and gastric cancer risk in the Shanghai Women's Health Study. Br J Cancer , 2012, 106(3): 585-591. [72] Neale RE, Clark PJ, Fawcett J, Fritschi L, Nagler BN, Risch HA, Walters RJ, Crawford WJ, Webb PM, Whiteman DC, Buchanan DD. Association between hypermethylation of DNA repetitive elements in white blood cell DNA and pancreatic cancer. Cancer Epidemiol , 2014, 38(5): 576-582. [73] Patchsung M, Boonla C, Amnattrakul P, Dissayabutra T, Mutirangura A, Tosukhowong P. Long interspersed nuclear element-1 hypomethylation and oxidative stress: correlation and bladder cancer diagnostic potential. PLoS One , 2012, 7(5): e37009. |
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