IGF2基因表达调控及其遗传变异在动物生长发育中的研究进展

doi:10.16431/j.cnki.1671-7236.2020.06.024

Abstract

Abstract: Insulin-like growth factor 2(IGF2),one of the insulin-like hormone family,widely involved in many physiological and metabolic processes of the body,which has an important role in cancer,neuroregulation,metabolic disease,osteoporosis,muscle development and lipidosis.IGF2 functions mainly through competitive binding to the IGF1R,IGF2R or IGF2BPs.Base on the IGF2 plays an important role in musle develop and lipidosis,it makes sense to mine molecular markers of IGF2.Furthermore,the internal regulation mechanism of IGF2 in livestock production was further analyzed.It was found that there was a significant correlation between the genetic variation of IGF2 gene and animal development.Genetic variation functions by affecting IGF2 gene imprinting,methylation,transcription factors binding or post-transcriptional regulation due to miRNA targeting binding.Therefore,this paper focused on the research progress of IGF2 gene expression regulation,gene imprinting,and epigenetic regulation about miRNA,in order to provide relevant reference for the study of IGF2 gene in the regulation of animal growth and development,and provide effective clues for molecular breeding in animal husbandry.

Key words: IGF2 gene; growth and development; transcription factor; heritable variation

CLC Number:

S811.3

ZHAO Haidong, WU Mingli, CHEN Pingbo, WEI Yanpei, WANG Shuhui, SUN Xiuzhu. Research Progress of IGF2 Gene Expression Regulation and Its Genetic Variation in Animal Growth and Development[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1844-1852.

References

[1] MOURMOURAS N,PHILIPPOU A,CHRISTOPOULOS P,et al.Differential expression of IGF-Ⅰ transcripts in bladder cancer[J].Anticancer Research,2018,38(6):3453-3459.
[2] BELLA P,FARINI A,BANFI S,et al.Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy[J].EMBO Molecular Medicine,2019,2:e11019.
[3] GARRETT S M,HSU E,THOMAS J M,et al.Insulin-like growth factor (IGF)-Ⅱ-mediated fibrosis in pathogenic lung conditions[J].PLoS One,2019,14(11):e0225422.
[4] LPEZ-BUESA P,BURGOS C,GALVE A,et al.Joint analysis of additive,dominant and first-order epistatic effects of four genes (IGF2,MC4R,PRKAG3 and LEPR) with known effects on fat content and fat distribution in pigs[J].Animal Genetics,2014,45(1):133-137.
[5] YU W,CUI X,WAN Z,et al.Silencing forkhead box M1 promotes apoptosis and autophagy through SIRT7/mTOR/IGF2 pathway in gastric cancer cells[J].Journal of Cellular Biochemistry,2018,119(11):9090-9098.
[6] MOSCHOS S J,MANTZOROS C S.The role of the IGF system in cancer:From basic to clinical studies and clinical applications[J].Oncology,2002,63(4):317-332.
[7] YU H,ROHA T.Role of the insulin-like growth factor family in cancer development and progression[J].Journal of the National Cancer Institute,2000,92(18):1472-1489.
[8] ROSENTHAL S M,BROWN E J,BRUNETTI A,et al.Fibroblast growth factor inhibits insulin-like growth factor-Ⅱ (IGF-Ⅱ gene expression and increases IGF-Ⅰ receptor abundance in BC3H-1 muscle cells[J].Molecular Endocrinology,1991,5(5):678-684.
[9] RUBINI M,WERNER H,GANDINI E,et al.Platelet-derived growth factor increases the activity of the promoter of the insulin-like growth factor-1(IGF-1) receptor gene[J].Experimental Cell Research,1994,211(2):374-379.
[10] WANG X,CAO X,DONG D,et al.Circular RNA TTN acts as a miR-432 sponge to facilitate proliferation and differentiation of myoblasts via the IGF2/PI3K/AKT signaling pathway[J].Molecular Therapy-Nucleic Acids,2019,18(12):966-980.
[11] SHAPIRO M R,WASSERFALL C H,MCGRAIL S M,et al.Insulin-like growth factor dysregulation both preceding and following type 1 diabetes diagnosis[J].Diabetes,2020,69(3):413-423.
[12] CHEN X,ZHANG R,ZHANG Q,et al.Microtia patients:Auricular chondrocyte ECM is promoted by CGF through IGF-1 activation of the IGF-1R/PI3K/AKT pathway[J].Journal of Cellular Physiology,2019,234(12):21817-21824.
[13] TIAN B Q,ZHAO Y M,LIANG T,et al.Curcumin inhibits urothelial tumor development by suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway[J].Journal of Drug Targeting,2017,25(7):1-24.
[14] DENLEY A,WALLACE J,COSGROVE L,et al.The insulin receptor isoform exon 11-(IR-A) in cancer and other diseases:A review[J].Hormone and Metabolic Research,2003,35(11/12):778-785.
[15] YI T,WANG T,SHI Y,et al.Long noncoding RNA 91H overexpression contributes to the growth and metastasis of HCC by epigenetically positively regulating IGF2 expression[J].Liver International,2020,40(2):456-467.
[16] WEIS F,MENTING J G,MARGETTS M B,et al.The signalling conformation of the insulin receptor ectodomain[J].Nature Communication,2018,9(1):4420.
[17] BHARATI J,RAMIN M,KRISTINA K,et al.RNA-Seq in 296 phased trios provides a high-resolution map of genomic imprinting[J].BMC Biology,2019,17(1):50.
[18] PARK K S,MITRA A,RAHAT B,et al.Loss of imprinting mutations define both distinct and overlapping roles for misexpression of IGF2 and of H19 lncRNA[J].Nucleic Acids Research,2017,22:12766-12779.
[19] ZHENG Q F,XU B,WANG H M,et al.Epigenetic alterations contribute to promoter activity of imprinting gene IGF2[J].Biochimica et Biophysica Acta-Gene Regulatory Mechanisms,2018,1861(2):117-124.
[20] SONG M A,ERNST T,TIIRIKAINEN M,et al.Methylation of imprinted IGF2 regions is associated with total,visceral,and hepatic adiposity in postmenopausal women[J].Epigenetics,2018,13(8):858-865.
[21] ÁCS O,PÉTERFIA B,HOLLÓSI P,et al.Methylation status of CYP27B1 and IGF2 correlate to BMI SDS in children with obesity[J].Obesity Facts,2017,10(4):353-362.
[22] PAUWELS S,GHOSH M,DUCA R C,et al.Maternal intake of methyl-group donors affects DNA methylation of metabolic genes in infants[J].Clinical Epigenetics,2017,9:16.
[23] NI W,PAN C,PAN Q,et al.Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage[J].Andrologia,2019,51(5):e13239.
[24] TSERGA A,BINDER A M,MICHELS K B.Impact of folic acid intake during pregnancy on genomic imprinting of IGF2/H19 and 1-carbon metabolism[J].Federation of American Societies for Experimental Biology Journal,2017,31(12):5149-5158.
[25] YI F,SHANG Y Q,LI B,et al.MicroRNA-193-5p modulates angiogenesis through IGF2 in type 2 diabetic cardiomyopathy[J].Biochemical & Biophysical Research Communications,2017,491(4):876-882.
[26] LIU M,ROTH A,YU M,et al.The IGF2 intronic miR-483 selectively enhances transcription from IGF2 fetal promoters and enhances tumorigenesis[J].Genes Development,2013,27(23):2543-2548.
[27] SONG C,YANG Z,DONG D,et al.miR-483 inhibits bovine myoblast cell proliferation and differentiation via IGF1/PI3K/AKT signal pathway[J].Journal of Cellular Physiology,2019,234(6):9839-9848.
[28] MARKLJUNG E,JIANG L,JAFFE J D,et al.ZBED6,a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth[J].PloS Biology,2009,7(12):e1000256.
[29] WANG X,JIANG L,WALLERMAN O,et al.ZBED6 negatively regulates insulin production,neuronal differentiation,and cell aggregation in MIN6 cells[J].FASEB Journal,2019,33(1):88-100.
[30] WANG X,JIANG L,WALLREMAN O,et al.Transcription factor ZBED6 affects gene expression,proliferation,and cell death in pancreatic beta cells[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(40):15997-16002.
[31] LIU X,LIU H,WANG M,et al.Disruption of the ZBED6 binding site in intron 3 of IGF2 by CRISPR/Cas9 leads to enhanced muscle development in Liang Guang Small Spotted pigs[J].Transgenic Research,2019,28(1):141-150.
[32] 王丹丹,唐雨婷,马月辉,等.用ZBED6基因敲除猪研究心脏发育的分子机制[J].中国农业科学,2018,51(7):1390-1400. WANG D D,TANG Y T,MA Y H,et al.Studying the molecular mechanism of heart development by using ZBED6 gene knockout pig[J].Scientia Agricultura Sinica,2018,51(7):1390-1400.(in Chinese)
[33] YOUNIS S,MILENA S,JULIE M,et al.The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals[J].Proceedings of the National Academy of Sciences,2018,115(9):E2048-E2057.
[34] HUANG Y Z,ZHANG L Z,LAI X S,et al.Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoter activity and DNA methylation in myoblasts[J].Scientific Reports,2014,4:4570.
[35] BELL A C,FELSENFELD G.Methylation of a CTCF-dependent boundary controls imprinted expression of the IGF2 gene[J].Nature,2000,405(6785):482-485.
[36] BUSSLINGER G A,STOCSITS R R,VAN P,et al.Cohesin is positioned in mammalian genomes by transcription,CTCF and Wapl[J].Nature,2017,544(7651):503-507.
[37] HUANG Z,WEN Y,SIMEL L,et al.CTCF binding at a novel intragenic binding site is associated with elevated IGF2 expression in serous epithelial ovarian cancer[J].Cancer Research,2009,69:5148.
[38] 田俊良.靶向修饰H19/IGF2的CTCF结合位点DNA甲基化对小鼠胚胎干细胞及早期胚胎发育的影响[D].呼和浩特:内蒙古大学,2019. TIAN J L.Effect of specific modification the DNA methylation at CTCF binding sites of H19/IGF2 locus on mouse embryonic stem cells and early embryo[D].Hohhot:Inner Mongolia University,2019.(in Chinese)
[39] QUENNEVILLE S,VERDE G,CORSINOTTI A,et al.In embryonic stem cells,ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions[J].Molecular Cell,2011,44(3):341-342.
[40] TADA Y,YAMAGUCHI Y,KINJO T,et al.The stem cell transcription factor ZFP57 induces IGF2 expression to promote anchorage-independent growth in cancer cells[J].Oncogene,2015,34(6):752-760.
[41] LUI J C,BARON J.Evidence that Igf2 down-regulation in postnatal tissues and up-regulation in malignancies is driven by transcription factor E2f3[J].Proceedings of the National Academy of Sciences,2013,110(15):6181-6186.
[42] YANG P,WANG Y,HOANG D,et al.A placental growth factor is silenced in mouse embryos by the zinc finger protein ZFP568[J].Science,2017,356(6339):757-759.
[43] SCHAGDARSURENGIN U,LAMMERT A,SCHUNK N,et al.Impairment of IGF2 gene expression in prostate cancer is triggered by epigenetic dysregulation of IGF2-DMR0 and its interaction with KLF4[J].Cell Communication and Signaling,2017,15(1):40-54.
[44] ERHARDT S,LYKO F,AINSCOUGH F X,et al.Polycomb-group proteins are involved in silencing processes caused by a transgenic element from the murine imprinted H19/Igf2 region in Drosophila[J].Development Genes & Evolution,2003,213(7):336-344.
[45] THORVALDSEN J L,DURAN K L,BARTOLOMEI M S.Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and IGF2[J].Genes & Development,1998,12(23):3693-3702.
[46] 洪雁,赵梅.miR-339通过靶向调控IGF2BP1抑制肺动脉平滑肌细胞的增殖[J].中国病理生理杂志,2018,34(10):1848-1854. HONG Y,ZHAO M.miR-339 suppresses proliferation of pulmonary arterial smooth muscle cells via regulating IGF2BP1[J].Chinese Journal of Pathophysiology,2018,34(10):1848-1854.(in Chinese)
[47] YANG Q,WANG P,DU X,et al.Direct repression of IGF2 is implicated in the anti-angiogenic function of microRNA-210 in human retinal endothelial cells[J].Angiogenesis,2018,21(2)313-323.
[48] 武建亮,徐利,卢纪和,等.猪IGF2基因多态性研究[J].猪业科学,2017,34(2):110-112. WU J L,XU L,LU J H,et al.Polymorphism of pig IGF2 gene[J].Pig Science,2017,34(2):110-112.(in Chinese)
[49] DIKKES P,JAFFE D B,GUO W H,et al.IGF2 knockout mice are resistant to kainic acid-induced seizures and neurodegeneration[J].Brain Research,2007,1175:85-95.
[50] UCHIMURA T,HOLLANDER J M,NAKAMURA D S,et al.An essential role for IGF2 in cartilage development and glucose metabolism during postnatal long bone growth[J].Development,2017,144(19):3533-3546.
[51] STRINGER J M,SUZUKI S,PASK A J,et al.Promoter-specific expression and imprint status of marsupial IGF2[J].PloS One,2012,7(7):e41690.
[52] HYUN S W,KIM S J,PARK K,et al.Characterization of the P4 promoter region of the human insulin-like growth factor Ⅱ gene[J].Federation of European Biochemical Societies Letters,1993,332(1-2):153-158.
[53] MASUNAGA Y,INOUE T,YAMOTO K,et al.IGF2 mutations[J].The Journal of Clinical Endocrinology & Metabolism,2020,105(1):dgz034.
[54] CRIADO L,BALLESTER M,CRESPO-PIAZUELO D,et al.Analysis of porcine IGF2 gene expression in adipose tissue and its effect on fatty acid composition[J].PloS One,2019,14(8):e0220708.
[55] 王彬彬,汪涵,马翔,等.苏淮猪肌内脂肪表型测定及其与IGF2和SCD基因的多态性关联性分析[J].畜牧与兽医,2018,50(3):20-25. WANG B B,WANG H,MA X,et al.Analysis of intramuscular fat content in Suhuai pigs and its correlation with polymorphisms of IGF2 and SCD genes[J].Animal Husbandry & Veterinary Medicine,2018,50(3):20-25.(in Chinese)
[56] 薛慧良,梁孝东.猪IGF2基因启动子区的遗传多态性分析[J].曲阜师范大学学报(自然科学版),2008,34(1):96-99. XUE H L,LIANG X D.Genetic polymorphisms of the porcine IGF2 gene in promoter[J].Journal of Qufu Normal University(Natural Science),2008,34(1):96-99.(in Chinese)
[57] HOU G,WANG D,GUAN S,et al.Associated analysis of single nucleotide polymorphisms of IGF2 gene's exon 8 with growth traits in Wuzhishan pig[J].Molecular Biology Reports,2010,37(1):497-500.
[58] 汪琦,柴志欣,钟金城.西藏牦牛IGF2基因内含子8遗传多态性及其遗传效应分析[J].西南农业学报,2016,29(8):1998-2003. WANG Q,CHAI Z X,ZHONG J C.Genetic polymorphisms and genetic effect of IGF2 gene intron 8 in Tibet yak[J].Southwest China Journal of Agricultural Sciences,2016,29(8):1998-2003.(in Chinese)
[59] 牛伟萍,刘晶,张金玉等.草原红牛IGF2基因外显子4的遗传多态性及遗传效应分析[J].吉林农业大学学报,2011,33(3):324-326. NIU W P,LIU J,ZHANG J Y,et al.Genetic polymorphisms in exon 4 of IGF2 gene of red cattle and its genetic effects[J].Journal of Jilin Agricultural University,2011,33(3):324-326.(in Chinese)
[60] 韩瑞华,昝林森,杨大鹏,等.秦川牛IGF2基因SNPs检测及其与胴体、肉质性状的相关性[J].遗传,2008,30(12):1579-1584. HAN R H,ZAN L S,YANG D P,et al.SNPs detection of IGF2 gene and its relationship with carcass and meat quality traits in Qinchuan cattle[J].Hereditas,2008,30(12):1579-1584.(in Chinese)
[61] ABO H G,EL-MAGD M A,EL-NAHAS A F,et al.Association of a novel SNP in exon 10 of the IGF2 gene with growth traits in Egyptian water buffalo (Bubalus bubalis)[J].Tropical Animal Health & Production,2014,46(6):947-952.
[62] HUANG Y Z,ZHAN Z Y,LI X Y,et al.SNP and haplotype analysis reveal IGF2 variants associated with growth traits in Chinese Qinchuan cattle[J].Molecular Biology Reports,2014,41(2):591-598.
[63] GOODALL J J,SCHMUTZ S M.IGF2 gene characterization and association with rib eye area in beef cattle[J].Animal Genetics,2007,38(2):154-161.
[64] 陈则东,沈晓鹏,穆洪云.鸡IGF2基因外显子1的多态性及其与生产性能的相关性[J].江苏农业科学,2016,44(6):331-332. CHEN Z D,SHEN X P,MU H Y.Polymorphism of exon 1 of chicken IGF2 gene and its correlation with production performance[J].Jiangsu Agricultural Sciences,2016,44(6):331-332.(in Chinese)
[65] 郭发荣,陈彬,李太山,等.鸡IGF2基因多态性及蛋白相关分析[J].中国畜牧杂志,2015,51(17):16-22. GUO F R,CHEN B,LI T S,et al.Analysis on oolymorphisms of IGF2 gene and the protein in chicken[J].Chinese Journal of Animal Science,2015,51(17):16-22.(in Chinese)
[66] YE Q,XU J G,GAO,X F,et al.Associations of IGF2 and DRD2 polymorphisms with laying traits in Muscovy duck[J].The Journal of Life and Environmental Sciences,2017,5(Suppl 1):e4083.
[67] 陈雪峰,杨国梁,俞菊华,等.吉富罗非鱼IGF2基因分离及其单核苷酸多态性与体型、增重相关性[J].动物学杂志,2010,45(2):107-114. CHEN X F,YANG G L,YU J H,et al.Isolation of IGF2 gene and correlation of its SNPs with fish sharp and weight gain in GIFT strain Nile Tilapia oreochromis niloticus[J].Chinese Journal of Zoology,2010,45(2):107-114.(in Chinese)
[68] 彭娜,曾丹,王晓清,等.中华鳖IGF2基因SNP标记与生长性状的关联分析[J].湖南农业大学学报(自然科学版),2018,44(1):88-94. PENG N,ZENG D,WANG X Q,et al.SNP markers on IGF2 gene in Pelodiscus sinensis and its correlation analysis to growth traits[J].Journal of Hunan Agricultural University(Natural Sciences),2018,44(1):88-94.(in Chinese)

Research Progress of IGF2 Gene Expression Regulation and Its Genetic Variation in Animal Growth and Development

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