多囊卵巢综合征易感基因dennd1a在斑马鱼模型中的表达特性
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摘要
目的探讨多囊卵巢综合征(PCOS)易感基因dennd1a在斑马鱼模型胚胎发育过程以及成鱼不同器官中的表达,构建适用于PCOS分子机制研究的斑马鱼实验动物模型。方法采用反转录-聚合酶链反应(RT-PCR)及实时荧光定量逆转录聚合酶链反应(qRT-PCR)技术,检测dennd1a基因在斑马鱼中的表达特性。利用胚胎整体原位杂交(WISH)方法,观察dennd1a基因在斑马鱼胚胎内的表达及分布。结果 qRT-PCR结果显示斑马鱼dennd1a mRNA的表达水平随着胚胎的发育而增加,且在多个组织中均有表达,于神经系统和雄性生殖系统中存在高表达。WISH结果表明该基因早期主要在神经系统表达。结论斑马鱼dennd1a基因的表达在胚胎早期发育中发挥重要作用,基因高表达尤其发现于中枢神经系统和雄性生殖系统,提示该基因的表达可能通过影响神经内分泌功能,调控雄激素的合成或释放,诱发PCOS并导致相关表型。
Objective To study the expression characteristics of polycystic ovary syndrome( PCOS) susceptibility gene dennd1a in developing embryos and in adult organs of zebrafish,and to establish zebrafish models for studies of the mechanisms underlying PCOS. Methods Using reverse transcription-polymerase chain reaction( RT-PCR) and realtime fluorescence quantitative reverse transcription polymerase chain reaction( qRT-PCR) techniques,we detected the expression of dennd1a in zebrafish. We also determined the expression pattern of dennd1a gene in zebrafish embryos using whole-mount in situ hybridization( WISH) assays. Results In embryos and juveniles,the expression level of dennd1a increased with age,and in adult animals,the expression was found in different organs. qRT-PCR and WISH showed the expression of dennd1a especially in the nervous system and male reproductive system. Conclusion In early stages of zebrafish development,high expression of dennd1a was found in the nervous system and male reproductive system,indicating this gene may regulate the development and function of the endocrine system and interrupt androgen synthesis and release,thereby inducing PCOS.
Objective To study the expression characteristics of polycystic ovary syndrome( PCOS) susceptibility gene dennd1a in developing embryos and in adult organs of zebrafish,and to establish zebrafish models for studies of the mechanisms underlying PCOS. Methods Using reverse transcription-polymerase chain reaction( RT-PCR) and realtime fluorescence quantitative reverse transcription polymerase chain reaction( qRT-PCR) techniques,we detected the expression of dennd1a in zebrafish. We also determined the expression pattern of dennd1a gene in zebrafish embryos using whole-mount in situ hybridization( WISH) assays. Results In embryos and juveniles,the expression level of dennd1a increased with age,and in adult animals,the expression was found in different organs. qRT-PCR and WISH showed the expression of dennd1a especially in the nervous system and male reproductive system. Conclusion In early stages of zebrafish development,high expression of dennd1a was found in the nervous system and male reproductive system,indicating this gene may regulate the development and function of the endocrine system and interrupt androgen synthesis and release,thereby inducing PCOS.
引文
[1]Li R,Zhang Q,Yang D,et al. Prevalence of polycystic ovary syndrome in women in China:a large communitybased study[J]. Hum Reprod,2013,28(9):2562-2569.
[2] Sirmans SM,Pate KA. Epidemiology,diagnosis,and management of polycystic ovary syndrome[J]. Clin Epidemiol,2013,6:1-13. doi:10.2147/CLEP.S37559.
[3]Chen ZJ,Zhao H,He L,et al. Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3,2p21 and 9q33.3[J]. Nat Genet,2011,43(1):55-59.
[4]Mcallister JM,Legro RS,Modi BP,et al. Functional genomics of PCOS:from GWAS to molecular mechanisms[J]. Trends Endocrinol Metab,2015,26(3):118-124.
[5] Cauvin C,Rosendale M,Gupta-Rossi N,et al. Rab35GTPase triggers switch-like recruitment of the lowe syndrome lipid phosphatase OCRL on newborn endosomes[J]. Curr Biol,2016,26(1):120-128.
[6]Goodarzi MO,Jones MR,Li X,et al. Replication of association of DENND1A and THADA variants with polycystic ovary syndrome in European cohorts[J]. J Med Genet,2012,49(2):90-95.
[7] Udhane SS,Fluck CE. Regulation of human(adrenal)androgen biosynthesis-Newinsights from novel throughput technology studies[J]. Biochem Pharmacol,2016,102:20-33. doi:10.1016/j.bcp.2015.10.010.
[8]孙梅,盛燕,马增香,等.多囊卵巢综合征易感基因与代谢表型相关性分析[J].中华妇产科杂志,2014,49(6):441-445.SUN Mei,SHENG Yan,MA Zengxiang,et al. Correlation analysis between polycystic ovary syndrome susceptibility genes and metabolic phenotypes[J]. Chinese Journal of Obstetrics and Gynecology,2014,49(6):441-445.
[9] Dunaif A. Perspectives in polycystic ovary syndrome:from hair to eternity[J]. J Clin Endocrinol Metab,2016,101(3):759-768.
[10]Zhao H,Xu X,Xing X,et al. Family-based analysis of susceptibility loci for polycystic ovary syndrome on chromosome 2p16. 3,2p21 and 9q33. 3[J]. Hum Reprod,2012,27(1):294-298.
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[12] Bao S, Cai JH, Yang SY, et al. Association of DENND1A gene polymorphisms with polycystic ovary syndrome:a meta-analysis[J]. J Clin Res Pediatr Endocrinol,2016,8(2):135-143.
[13]Rinkwitz S,Mourrain P,Becker TS. Zebrafish:an integrative system for neurogenomics and neurosciences[J].Prog Neurobiol,2011,93(2):231-243.
[14] Iwanami N. Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency[J]. Exp Hematol,2014,42(8):697-706.
[15]Hoo JY,Kumari Y,Shaikh MF,et al. Zebrafish:a versatile animal model for fertility research[J]. Biomed Res Int,2016,2016:9732780. doi:10.1155/2016/9732780.
[16]Yildiz BO,Bozdag G,Yapici Z,et al. Prevalence,phenotype and cardiometabolic risk of polycystic ovary syndrome under different diagnostic criteria[J]. Hum Reprod,2012,27(10):3067-3073.
[17]李婧博,刘洪彬,贾月月,等. microRNA-183在PCOS胰岛素抵抗中的表达及其临床意义[J].山东大学学报(医学版),2017,55(1):69-74.LI Jingbo,LIU Hongbin,JIA Yueyue,et al. Expression and clinical significance of microRNA-183 in polycystic ovary syndrome with insulin resistance[J]. Journal of Shandong University(Health Sciences),2017,55(1):69-74.
[18]Chen L,Hu LM,Wang YF,et al. Genome-wide association study for SNPs associated with PCOS in human patients[J]. Exp Ther Med,2017,14(5):4896-4900.
[19]Ha L,Shi Y,Zhao J,et al. Association Study between Polycystic Ovarian Syndrome and the Susceptibility Genes Polymorphisms in Hui Chinese Women[J]. PLoS One,2015,10(5):e126505. doi:10. 1371/journal.pone.0126505.
[20]Gammoh E,Arekat MR,Saldhana FL,et al. DENND1A gene variants in Bahraini Arab women with polycystic ovary syndrome[J]. Gene,2015,560(1):30-33.
[21] Saxena R,Georgopoulos NA,Braaten TJ,et al. Han Chinese polycystic ovary syndrome risk variants in women of European ancestry:relationship to FSH levels and glucose tolerance[J]. Hum Reprod,2015,30(6):1454-1459.
[22]彭丹红,罗嘉莉,凌丽,等. DENND1A基因多态性与多囊卵巢综合征易感性的Meta分析[J].南京医科大学学报(自然科学版),2017,37(10):1362-1368.PENG Danhong,LUO Jiali,LING Li,et al. Meta-analysis on relationship between DENND1A polymorphisms and polycystic ovary syndrome[J]. Journal of Nanjing Medical University(Natural Sciences),2017,37(10):1362-1368.
[23]Dallel M,Sarray S,Douma Z,et al. Differential association of DENND1A genetic variants with polycystic ovary syndrome in Tunisian but not Bahraini Arab women[J]. Gene,2018,647:79-84. doi:10. 1016/j. gene.2018.01.028.
[24]Tee MK,Speek M,Legeza B,et al. Alternative splicing of DENND1A,a PCOS candidate gene,generates variant 2[J]. Mol Cell Endocrinol,2016,434:25-35. doi:10.1016/j.mce.2016.06.011.
[25] Mcallister JM,Modi B,Miller BA,et al. Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype[J]. Proc Natl Acad Sci U S A,2014,111(15):E1519-E1527.
[26] Cui L,Li G,Zhong W,et al. Polycystic ovary syndrome susceptibility single nucleotide polymorphisms in women with a single PCOS clinical feature[J]. Hum Reprod,2015,30(3):732-736.
[27]Wang Z,Li T,Zhang W,et al. Variants in DENND1A and LHCGR are associated with endometrioid adenocarcinoma[J]. Gynecol Oncol,2012,127(2):403-405.
[28] Lee AP,Brenner S,Venkatesh B. Mouse transgenesis identifies conserved functional enhancers and cis-regulatory motif in the vertebrate LIMhomeobox gene Lhx2 locus[J]. PLoS One,2011,6(5):e20088. doi:10.1371/journal.pone.0020088.
[29] Marat AL,Mcpherson PS. The connecdenn family,Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery[J]. J Biol Chem,2010,285(14):10627-10637. doi:0.1074/jbc.M109.050930.
[30]Kent SC,Gnatuk CL. Hyperandrogenism and hyperinsulinism in children of women with polycystic ovary syndrome:a controlled study[J]. J Clin Endocrinol Metab,2008,93(5):1662-1669.
[31] Leibel NI,Baumann EE. Relationship of adolescent polycystic ovary syndrome to parental metabolic syndrome[J]. J Clin Endocrinol Metab,2006,91(4):1275-1283.
[32] Recabarren SE,Smith R. Metabolic profile in sons of women with polycystic ovary syndrome[J]. J Clin Endocrinol Metab,2008,93(5):1820-1826.
[33] Torvinen A,Koivunen R. Metabolic and reproductive characteristics of first-degree relatives of women with self-reported oligo-amenorrhoea and hirsutism[J]. Gynecol Endocrinol,2011,27(9):630-635.
[2] Sirmans SM,Pate KA. Epidemiology,diagnosis,and management of polycystic ovary syndrome[J]. Clin Epidemiol,2013,6:1-13. doi:10.2147/CLEP.S37559.
[3]Chen ZJ,Zhao H,He L,et al. Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3,2p21 and 9q33.3[J]. Nat Genet,2011,43(1):55-59.
[4]Mcallister JM,Legro RS,Modi BP,et al. Functional genomics of PCOS:from GWAS to molecular mechanisms[J]. Trends Endocrinol Metab,2015,26(3):118-124.
[5] Cauvin C,Rosendale M,Gupta-Rossi N,et al. Rab35GTPase triggers switch-like recruitment of the lowe syndrome lipid phosphatase OCRL on newborn endosomes[J]. Curr Biol,2016,26(1):120-128.
[6]Goodarzi MO,Jones MR,Li X,et al. Replication of association of DENND1A and THADA variants with polycystic ovary syndrome in European cohorts[J]. J Med Genet,2012,49(2):90-95.
[7] Udhane SS,Fluck CE. Regulation of human(adrenal)androgen biosynthesis-Newinsights from novel throughput technology studies[J]. Biochem Pharmacol,2016,102:20-33. doi:10.1016/j.bcp.2015.10.010.
[8]孙梅,盛燕,马增香,等.多囊卵巢综合征易感基因与代谢表型相关性分析[J].中华妇产科杂志,2014,49(6):441-445.SUN Mei,SHENG Yan,MA Zengxiang,et al. Correlation analysis between polycystic ovary syndrome susceptibility genes and metabolic phenotypes[J]. Chinese Journal of Obstetrics and Gynecology,2014,49(6):441-445.
[9] Dunaif A. Perspectives in polycystic ovary syndrome:from hair to eternity[J]. J Clin Endocrinol Metab,2016,101(3):759-768.
[10]Zhao H,Xu X,Xing X,et al. Family-based analysis of susceptibility loci for polycystic ovary syndrome on chromosome 2p16. 3,2p21 and 9q33. 3[J]. Hum Reprod,2012,27(1):294-298.
[11] Eriksen MB,Nielsen MF,Brusgaard K,et al. Genetic alterations within the DENND1A gene in patients with polycystic ovary syndrome(PCOS)[J]. PLoS One,2013,8(9):e77186. doi:10. 1371/journal. pone.0077186.
[12] Bao S, Cai JH, Yang SY, et al. Association of DENND1A gene polymorphisms with polycystic ovary syndrome:a meta-analysis[J]. J Clin Res Pediatr Endocrinol,2016,8(2):135-143.
[13]Rinkwitz S,Mourrain P,Becker TS. Zebrafish:an integrative system for neurogenomics and neurosciences[J].Prog Neurobiol,2011,93(2):231-243.
[14] Iwanami N. Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency[J]. Exp Hematol,2014,42(8):697-706.
[15]Hoo JY,Kumari Y,Shaikh MF,et al. Zebrafish:a versatile animal model for fertility research[J]. Biomed Res Int,2016,2016:9732780. doi:10.1155/2016/9732780.
[16]Yildiz BO,Bozdag G,Yapici Z,et al. Prevalence,phenotype and cardiometabolic risk of polycystic ovary syndrome under different diagnostic criteria[J]. Hum Reprod,2012,27(10):3067-3073.
[17]李婧博,刘洪彬,贾月月,等. microRNA-183在PCOS胰岛素抵抗中的表达及其临床意义[J].山东大学学报(医学版),2017,55(1):69-74.LI Jingbo,LIU Hongbin,JIA Yueyue,et al. Expression and clinical significance of microRNA-183 in polycystic ovary syndrome with insulin resistance[J]. Journal of Shandong University(Health Sciences),2017,55(1):69-74.
[18]Chen L,Hu LM,Wang YF,et al. Genome-wide association study for SNPs associated with PCOS in human patients[J]. Exp Ther Med,2017,14(5):4896-4900.
[19]Ha L,Shi Y,Zhao J,et al. Association Study between Polycystic Ovarian Syndrome and the Susceptibility Genes Polymorphisms in Hui Chinese Women[J]. PLoS One,2015,10(5):e126505. doi:10. 1371/journal.pone.0126505.
[20]Gammoh E,Arekat MR,Saldhana FL,et al. DENND1A gene variants in Bahraini Arab women with polycystic ovary syndrome[J]. Gene,2015,560(1):30-33.
[21] Saxena R,Georgopoulos NA,Braaten TJ,et al. Han Chinese polycystic ovary syndrome risk variants in women of European ancestry:relationship to FSH levels and glucose tolerance[J]. Hum Reprod,2015,30(6):1454-1459.
[22]彭丹红,罗嘉莉,凌丽,等. DENND1A基因多态性与多囊卵巢综合征易感性的Meta分析[J].南京医科大学学报(自然科学版),2017,37(10):1362-1368.PENG Danhong,LUO Jiali,LING Li,et al. Meta-analysis on relationship between DENND1A polymorphisms and polycystic ovary syndrome[J]. Journal of Nanjing Medical University(Natural Sciences),2017,37(10):1362-1368.
[23]Dallel M,Sarray S,Douma Z,et al. Differential association of DENND1A genetic variants with polycystic ovary syndrome in Tunisian but not Bahraini Arab women[J]. Gene,2018,647:79-84. doi:10. 1016/j. gene.2018.01.028.
[24]Tee MK,Speek M,Legeza B,et al. Alternative splicing of DENND1A,a PCOS candidate gene,generates variant 2[J]. Mol Cell Endocrinol,2016,434:25-35. doi:10.1016/j.mce.2016.06.011.
[25] Mcallister JM,Modi B,Miller BA,et al. Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype[J]. Proc Natl Acad Sci U S A,2014,111(15):E1519-E1527.
[26] Cui L,Li G,Zhong W,et al. Polycystic ovary syndrome susceptibility single nucleotide polymorphisms in women with a single PCOS clinical feature[J]. Hum Reprod,2015,30(3):732-736.
[27]Wang Z,Li T,Zhang W,et al. Variants in DENND1A and LHCGR are associated with endometrioid adenocarcinoma[J]. Gynecol Oncol,2012,127(2):403-405.
[28] Lee AP,Brenner S,Venkatesh B. Mouse transgenesis identifies conserved functional enhancers and cis-regulatory motif in the vertebrate LIMhomeobox gene Lhx2 locus[J]. PLoS One,2011,6(5):e20088. doi:10.1371/journal.pone.0020088.
[29] Marat AL,Mcpherson PS. The connecdenn family,Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery[J]. J Biol Chem,2010,285(14):10627-10637. doi:0.1074/jbc.M109.050930.
[30]Kent SC,Gnatuk CL. Hyperandrogenism and hyperinsulinism in children of women with polycystic ovary syndrome:a controlled study[J]. J Clin Endocrinol Metab,2008,93(5):1662-1669.
[31] Leibel NI,Baumann EE. Relationship of adolescent polycystic ovary syndrome to parental metabolic syndrome[J]. J Clin Endocrinol Metab,2006,91(4):1275-1283.
[32] Recabarren SE,Smith R. Metabolic profile in sons of women with polycystic ovary syndrome[J]. J Clin Endocrinol Metab,2008,93(5):1820-1826.
[33] Torvinen A,Koivunen R. Metabolic and reproductive characteristics of first-degree relatives of women with self-reported oligo-amenorrhoea and hirsutism[J]. Gynecol Endocrinol,2011,27(9):630-635.