脊椎动物雌激素对家蚕卵黄原蛋白基因表达的调控及其与蜕皮激素受体相互作用的MST分析
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摘要
【目的】雌激素雌二醇(E_2)在脊椎动物中通过雌激素受体(ER)调节脊椎动物的生长发育和繁殖。昆虫中并没有ER,但有研究显示某些昆虫能对E_2产生一定的应答。本研究在明确E_2能影响家蚕Bombyx mori卵黄原蛋白基因(BmVg)表达的前提下,分析其对这一基因表达的调控机制。【方法】取BmVg高量表达时的家蚕雌性幼虫(上蔟后60 h)脂肪体,用不同浓度(0.001,0.05,0.5,5和50 nmol/L)E_2处理后,通过qRT-PCR检测BmVg表达的变化,分析家蚕对E_2的应答;克隆家蚕蜕皮激素受体基因(BmEcRB1),构建原核表达载体,表达并纯化BmEcRB1蛋白后与E_2体外孵育,通过微量热泳动仪(MST)分析E_2与BmEcRB1的结合情况。【结果】不同浓度E_2均显著抑制离体培养的雌蚕脂肪体中BmVg基因的表达。MST实验证实,E_2与BmEcRB1具有一定的亲和力,解离常数(Kd)为77.8±22μmol/L。【结论】结果说明,E_2对BmVg表达的影响是通过与蜕皮激素(20E)竞争性结合BmEcRB1实现的。
【Aim】Estradiol( E_2),an estrogen hormone,regulates the growth and reproduction through estrogen receptors( ERs) in vertebrates. There is no ER in insects,but studies showed that some insects can respond to E_2. In this study,the possible regulatory mechanisms of the expression of vitellogenin gene by E_2 was elucidated in the silkworm,Bombyx mori,after confirming that E_2 influenced the expression of BmVg in female larvae of the silkworm. 【Methods 】The expression level of BmVg was detected in the fat body of female larvae(60 h after mounting) of B. mori after in vitro treatment with different concentrations of E_2(0. 001,0. 05,0. 5,5 and 50 nmol/L) by qRT-PCR,and the response of the silkworm to E_2 was analyzed. The ecdysone receptor gene of B. mori( BmEcRB1) was cloned and its prokaryotic expression vector was constructed. The BmEcRB1 protein was purified and incubated with E_2 in vitro,and the binding characteristics of E_2 to BmEcRB1 were assayed by microscale thermophoresis(MST). 【Results】Different concentrations of E_2 significantly inhibited the expression of BmVg of female larvae of B. mori after in vitro culture,and the inhibitory effect was more obvious with the increase of E_2 concentration. MST results showed that E_2 had a certain affinity with BmEcRB1,with the dissociation constant( Kd) value of 77. 8 ± 22 μmol/L. 【Conclusion 】 The results suggest that E_2 inhibits the expression of BmVg in the silkworm through competition with ecdysone to bind BmEcRB1.
【Aim】Estradiol( E_2),an estrogen hormone,regulates the growth and reproduction through estrogen receptors( ERs) in vertebrates. There is no ER in insects,but studies showed that some insects can respond to E_2. In this study,the possible regulatory mechanisms of the expression of vitellogenin gene by E_2 was elucidated in the silkworm,Bombyx mori,after confirming that E_2 influenced the expression of BmVg in female larvae of the silkworm. 【Methods 】The expression level of BmVg was detected in the fat body of female larvae(60 h after mounting) of B. mori after in vitro treatment with different concentrations of E_2(0. 001,0. 05,0. 5,5 and 50 nmol/L) by qRT-PCR,and the response of the silkworm to E_2 was analyzed. The ecdysone receptor gene of B. mori( BmEcRB1) was cloned and its prokaryotic expression vector was constructed. The BmEcRB1 protein was purified and incubated with E_2 in vitro,and the binding characteristics of E_2 to BmEcRB1 were assayed by microscale thermophoresis(MST). 【Results】Different concentrations of E_2 significantly inhibited the expression of BmVg of female larvae of B. mori after in vitro culture,and the inhibitory effect was more obvious with the increase of E_2 concentration. MST results showed that E_2 had a certain affinity with BmEcRB1,with the dissociation constant( Kd) value of 77. 8 ± 22 μmol/L. 【Conclusion 】 The results suggest that E_2 inhibits the expression of BmVg in the silkworm through competition with ecdysone to bind BmEcRB1.
引文
Amin ES,Bassiouny AR,1979.Estrone in Olea-europaea kernel.Phytochemistry,18(2):344.
Arukwe A,Goks?yr A,2003.Eggshell and egg yolk proteins in fish:hepatic proteins for the next generation:oogenetic,population,and evolutionary implications of endocrine disruption.Comp.Hepatol.,2(1):4.
Billas IM,Iwema T,Garnier JM,Mitschler A,Rochel N,Moras D,2003.Structural adaptability in the ligand-binding pocket of the ecdysone hormone receptor.Nature,426(6962):91-96.
Bradbrook DA,Clement CY,Cook B,Dinan L,1990.The occurrence of vertebrate-type steroids in insects and a comparison with ecdysteroid levels.Comp.Biochem.Physiol.B,95(2):365-374.
Das S,2016.Vertebrate hormones in insects:the role of estrogen in silkworm-a review.Turk.J.Zool.,40:297-302.
Denlinger DL,Brueggemeier RW,Mechoulam R,Katlic N,Yocum LB,Yocum GD,1986.Estrogens and androgens in insects.J.Cell.Biochem.,11(12):61.
Fujimoto Y,Ikekawa N,Ogiso M,Ohnishi E,1986.Characterization of17-beta-estradiol 3-(beta-D-glucopyranoside)and 17-(alpha-D-glucopyranoside)as the metabolites of 17-beta-estradiol in the cultured ovaries of the silkworm,Bombyx mori.Experientia,42(5):567-568.
Garcia-Alonso J,Hoeger U,Rebscher N,2006.Regulation of vitellogenesis in Nereis virens(Annelida:Polychaeta):effect of estradiol-17 beta on eleocytes.Comp.Biochem.Physiol.A,143(1):55-61.
Heldring N,Pike A,Andersson S,Matthews J,Cheng G,Hartman J,Tujague M,Strom A,Treuter E,Warner M,Gustafsson JA,2007.Estrogen receptors:how do they signal and what are their targets.Physiol.Rev.,87(3):905-931.
Hiruma K,Riddiford LM,2001.Regulation of transcription factors MHR4 andβFTZ-F1 by 20-hydroxyecdysone during a larval molt in the tobacco hornworm,Manduca sexta.Dev.Biol.,232(1):265-274.
Huang J,Li X,Yi P,Hilf R,Bambara RA,Muyan M,2004.Targeting estrogen responsive elements(EREs):design of potent transactivators for ERE-containing genes.Mol.Cell Endocrinol.,218(1-2):65-78.
Jerabek-Willemsen M,Wienken CJ,Braun D,Baaske P,Duhr S,2011.Molecularinteraction studies using microscale thermophoresis.Assay.Drug.Dev.Techn.,9(4):342-353.
Kamimura M,Tomita S,Kiuchi M,Fujiwara H,1997.Tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms ecdysteroid-dependent transcription in cultured anterior silk glands.Eur.J.Biochem.,248(3):786-793.
Kang IJ,Yokota H,Oshima Y,Tsuruda Y,Yamaguchi T,Maeda M,Imada N,Tadokoro H,Honjo T,2002.Effect of 17β-estradiol on the reproduction of Japanese medaka(Oryzias latipes).Chemosphere,47(1):71-80.
Keshan B,Ray AK,1998.Action of estradiol-17βon the synthetic activity of the silk gland in Bombyx mori L.J.Insect Physiol.,44(5):491-498.
Keshan B,Ray AK,2001.The presence of estradiol-17βand its specific binding sites in posterior silk gland of Bombyx mori.Gen.Comp.Endocr.,123(1):23-30.
Liu DX,Zhang ZP,Gladwell W,Teng CT,2003.Estrogen stimulates estrogen-related receptor alpha gene expression through conserved hormone response elements.Endocrinology,144(11):4894-4904.
Mao YX,Yu LL,Yang R,Qu LB,Harrington PD,2015.A novel method for the study of molecular interaction by using microscale thermophoresis.Talanta,132(132):894-901.
Martinez E,Givel F,Wahli W,1991.A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.EMBO J.,10(2):263-268.
Matozzo V,Gagne F,Marin MG,Ricciardi F,Blaise C,2008.Vitellogenin as a biomarker of exposure to estrogenic compounds in aquatic invertebrates:a review.Environ.Int.,34(4):531-545.
Mechoulam R,Brueggemeier RW,Denlinger DL,1984.Estrogens in insects.Experientia,40(9):942-944.
Novillo A,Won SJ,Li C,Callard IP,2005.Changes in nuclear receptor and vitellogenin gene expression in response to steroids and heavy metal in Caenorhabditis elegans.Integr.Comp.Biol.,45(1):61-71.
Oberdorster E,Clay MA,Cottam DM,Wilmot FA,Mc Lachlan JA,Milner MJ,2001.Common phytochemicals are ecdysteroid agonists and antagonists:a possible evolutionary link between vertebrate and invertebrate steroid hormones.J.Steroid Biochem.Mol.Biol.,77(4-5):229-238.
Ohnishi E,Ogiso M,Wakabayashi K,Fujimoto Y,Ikekawa N,1985.Identification of estradiol in the ovaries of the silkworm,Bombyx mori.Gen.Comp.Endocrinol.,60(1):35-38.
Rothschild M,Ford B,1964a.Breeding of rabbit flea(Spilopsyllus cuniculi(Dale))controlled by reproductive hormones of host.Nature,201(4914):103.
Rothschild M,Ford B,1964b.Matruation+egg-laying of rabbit flea.induced by external application of hydrocortisone.Nature,203(4941):210.
Roy S,De J,Kundu S,Biswas A,Pramanik M,Ray AK,2007.Estradiol-17β:tracing its metabolic significance in female fatbody of fifth instar larvae of silkworm,Bombyx mori L(race:Nistari).Life Sci.,80(5):446-453.
Seidel SAI,Dijkman PM,Lea WA,van den Bogaart G,JerabekWillemsen M,Lazic A,Joseph JS,Srinivasan P,Baaske P,Simeonov A,Katritch I,Melo FA,Ladbury JE,Schreiber G,Watts A,Braun D,Duhr S,2013.Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.Methods,59(3):301-315.
Shen G,Lin Y,Yang C,Xing R,Zhang H,Chen E,Han C,Liu H,Zhang W,Xia Q,2015.Vertebrate estrogen regulates the development of female characteristics in silkworm,Bombyx mori.Gen.Comp.Endocrinol.,210:30-37.
Shen GW,Wang JY,Yang CW,Zhang HY,Xing RM,Lin Y,Xia QY,2014.Do insects have vertebrate sex hormones?Acta Entomol.Sin.,57(11):1351-1359.[沈关望,王继岩,杨从文,张海燕,邢润苗,林英,夏庆友,2014.昆虫中存在高等动物性激素吗?昆虫学报,57(11):1351-1359]
Stanley-Samuelson DW,1987.Physiological roles of prostaglandins and other eicosanoids in invertebrates.Biol.Bull.,173(1):92-109.
Swetha CH,Girish BP,Reddy PS,2016.Elucidation of the role of estradiol and progesterone in regulating reproduction in the edible crab,Oziothelphusa senex senex.Rsc.Adv.,6(30):24959-24967.
Tada N,Nakao A,Hoshi H,Saka M,Kamata Y,2008.Vitellogenin,a biomarker for environmental estrogenic pollution,of Reeves'pond turtles:analysis of similarity for its amino acid sequence and cognate mRNA expression after exposure to estrogen.J.Vet.Med.Sci.,70(3):227-234.
Thornton JW,Need E,Crews D,2003.Resurrecting the ancestral steroid receptor:ancient origin of estrogen signaling.Science,301(5640):1714-1717.
Tominaga N,Ura K,Kawakami M,Kawaguchi T,Kohra S,Mitsui Y,Iguchi T,Arizono K,2003.Caenorhabditis elegans responses to specific steroid hormones.J.Heal.Sci.,49(1):28-33.
Tyler CR,vander Eerden B,Jobling S,Panter G,Sumpter JP,1996.Measurement of vitellogenin,a biomarker for exposure to oestrogenic chemicals,in a wide variety of cyprinid fish.J.Comp.Physiol.BBiochem.Sys.Environ.Physiol.,166(7):418-426.
Wienken CJ,Baaske P,Rothbauer U,Braun D,Duhr S,2010.Proteinbinding assays in biological liquids using microscale thermophoresis.Nat.Commun.,1(7):100.
Xu J,Wang YQ,Li ZQ,Ling L,Zeng BS,You L,Chen YZ,Aslam AF,Huang YP,Tan AJ,2014.Functional characterization of the vitellogenin promoter in the silkworm,Bombyx mori.Insect Mol.Biol.,23(5):550-557.
Yang C,Lin Y,Liu H,Shen G,Luo J,Zhang H,Peng Z,Chen E,Xing R,Han C,Xia Q,2014a.The Broad Complex isoform 2(Br C-Z2)transcriptional factor plays a critical role in vitellogenin transcription in the silkworm,Bombyx mori.Biochim.Biophys.Acta,1840(2014):2674-2684.
Yang CW,Lin Y,Shen GW,Chen EX,Wang YX,Luo J,Zhang HY,Xing RM,Xia QY,2014b.Female qualities in males:vitellogenin synthesis induced by ovary transplants into the male silkworm,Bombyx mori.Biochem.Biophys.Res.Commun.,453(1):31-36.
Yang HJ,Lee JW,Lee SH,Ryu JS,Kwak DH,Nam KS,Park YI,Lee YC,Jung KY,Choo YK,2010.Estrogenic activity produced by aqueous extracts of silkworm(Bombyx mori)pupae in ovariectomized rats.Am.J.Chin.Med.,38(1):89-97.
Yocum LB,Denlinger DL,Katlic NE,Brueggemeier RW,Mechoulam R,1987.A developmental profile of estrogen and androgen radioimmunoactive substances in the flesh fly,Sarcophaga crassipalpis.Insect Biochem.,17(8):1149-1153.
Zhao Y,Hu J,2012.Development of a molecular biomarker for detecting intersex after exposure of male medaka fish to synthetic estrogen.Environ.Toxicol.Chem.,31(8):1765-1773.
Zhao Y,Li Y,Lou G,Zhao L,Xu Z,Zhang Y,He F,2012.MiR-137targets estrogen-related receptor alpha and impairs the proliferative and migratory capacity of breast cancer cells.PLo S ONE,7(6):e39102.
Arukwe A,Goks?yr A,2003.Eggshell and egg yolk proteins in fish:hepatic proteins for the next generation:oogenetic,population,and evolutionary implications of endocrine disruption.Comp.Hepatol.,2(1):4.
Billas IM,Iwema T,Garnier JM,Mitschler A,Rochel N,Moras D,2003.Structural adaptability in the ligand-binding pocket of the ecdysone hormone receptor.Nature,426(6962):91-96.
Bradbrook DA,Clement CY,Cook B,Dinan L,1990.The occurrence of vertebrate-type steroids in insects and a comparison with ecdysteroid levels.Comp.Biochem.Physiol.B,95(2):365-374.
Das S,2016.Vertebrate hormones in insects:the role of estrogen in silkworm-a review.Turk.J.Zool.,40:297-302.
Denlinger DL,Brueggemeier RW,Mechoulam R,Katlic N,Yocum LB,Yocum GD,1986.Estrogens and androgens in insects.J.Cell.Biochem.,11(12):61.
Fujimoto Y,Ikekawa N,Ogiso M,Ohnishi E,1986.Characterization of17-beta-estradiol 3-(beta-D-glucopyranoside)and 17-(alpha-D-glucopyranoside)as the metabolites of 17-beta-estradiol in the cultured ovaries of the silkworm,Bombyx mori.Experientia,42(5):567-568.
Garcia-Alonso J,Hoeger U,Rebscher N,2006.Regulation of vitellogenesis in Nereis virens(Annelida:Polychaeta):effect of estradiol-17 beta on eleocytes.Comp.Biochem.Physiol.A,143(1):55-61.
Heldring N,Pike A,Andersson S,Matthews J,Cheng G,Hartman J,Tujague M,Strom A,Treuter E,Warner M,Gustafsson JA,2007.Estrogen receptors:how do they signal and what are their targets.Physiol.Rev.,87(3):905-931.
Hiruma K,Riddiford LM,2001.Regulation of transcription factors MHR4 andβFTZ-F1 by 20-hydroxyecdysone during a larval molt in the tobacco hornworm,Manduca sexta.Dev.Biol.,232(1):265-274.
Huang J,Li X,Yi P,Hilf R,Bambara RA,Muyan M,2004.Targeting estrogen responsive elements(EREs):design of potent transactivators for ERE-containing genes.Mol.Cell Endocrinol.,218(1-2):65-78.
Jerabek-Willemsen M,Wienken CJ,Braun D,Baaske P,Duhr S,2011.Molecularinteraction studies using microscale thermophoresis.Assay.Drug.Dev.Techn.,9(4):342-353.
Kamimura M,Tomita S,Kiuchi M,Fujiwara H,1997.Tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms ecdysteroid-dependent transcription in cultured anterior silk glands.Eur.J.Biochem.,248(3):786-793.
Kang IJ,Yokota H,Oshima Y,Tsuruda Y,Yamaguchi T,Maeda M,Imada N,Tadokoro H,Honjo T,2002.Effect of 17β-estradiol on the reproduction of Japanese medaka(Oryzias latipes).Chemosphere,47(1):71-80.
Keshan B,Ray AK,1998.Action of estradiol-17βon the synthetic activity of the silk gland in Bombyx mori L.J.Insect Physiol.,44(5):491-498.
Keshan B,Ray AK,2001.The presence of estradiol-17βand its specific binding sites in posterior silk gland of Bombyx mori.Gen.Comp.Endocr.,123(1):23-30.
Liu DX,Zhang ZP,Gladwell W,Teng CT,2003.Estrogen stimulates estrogen-related receptor alpha gene expression through conserved hormone response elements.Endocrinology,144(11):4894-4904.
Mao YX,Yu LL,Yang R,Qu LB,Harrington PD,2015.A novel method for the study of molecular interaction by using microscale thermophoresis.Talanta,132(132):894-901.
Martinez E,Givel F,Wahli W,1991.A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.EMBO J.,10(2):263-268.
Matozzo V,Gagne F,Marin MG,Ricciardi F,Blaise C,2008.Vitellogenin as a biomarker of exposure to estrogenic compounds in aquatic invertebrates:a review.Environ.Int.,34(4):531-545.
Mechoulam R,Brueggemeier RW,Denlinger DL,1984.Estrogens in insects.Experientia,40(9):942-944.
Novillo A,Won SJ,Li C,Callard IP,2005.Changes in nuclear receptor and vitellogenin gene expression in response to steroids and heavy metal in Caenorhabditis elegans.Integr.Comp.Biol.,45(1):61-71.
Oberdorster E,Clay MA,Cottam DM,Wilmot FA,Mc Lachlan JA,Milner MJ,2001.Common phytochemicals are ecdysteroid agonists and antagonists:a possible evolutionary link between vertebrate and invertebrate steroid hormones.J.Steroid Biochem.Mol.Biol.,77(4-5):229-238.
Ohnishi E,Ogiso M,Wakabayashi K,Fujimoto Y,Ikekawa N,1985.Identification of estradiol in the ovaries of the silkworm,Bombyx mori.Gen.Comp.Endocrinol.,60(1):35-38.
Rothschild M,Ford B,1964a.Breeding of rabbit flea(Spilopsyllus cuniculi(Dale))controlled by reproductive hormones of host.Nature,201(4914):103.
Rothschild M,Ford B,1964b.Matruation+egg-laying of rabbit flea.induced by external application of hydrocortisone.Nature,203(4941):210.
Roy S,De J,Kundu S,Biswas A,Pramanik M,Ray AK,2007.Estradiol-17β:tracing its metabolic significance in female fatbody of fifth instar larvae of silkworm,Bombyx mori L(race:Nistari).Life Sci.,80(5):446-453.
Seidel SAI,Dijkman PM,Lea WA,van den Bogaart G,JerabekWillemsen M,Lazic A,Joseph JS,Srinivasan P,Baaske P,Simeonov A,Katritch I,Melo FA,Ladbury JE,Schreiber G,Watts A,Braun D,Duhr S,2013.Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.Methods,59(3):301-315.
Shen G,Lin Y,Yang C,Xing R,Zhang H,Chen E,Han C,Liu H,Zhang W,Xia Q,2015.Vertebrate estrogen regulates the development of female characteristics in silkworm,Bombyx mori.Gen.Comp.Endocrinol.,210:30-37.
Shen GW,Wang JY,Yang CW,Zhang HY,Xing RM,Lin Y,Xia QY,2014.Do insects have vertebrate sex hormones?Acta Entomol.Sin.,57(11):1351-1359.[沈关望,王继岩,杨从文,张海燕,邢润苗,林英,夏庆友,2014.昆虫中存在高等动物性激素吗?昆虫学报,57(11):1351-1359]
Stanley-Samuelson DW,1987.Physiological roles of prostaglandins and other eicosanoids in invertebrates.Biol.Bull.,173(1):92-109.
Swetha CH,Girish BP,Reddy PS,2016.Elucidation of the role of estradiol and progesterone in regulating reproduction in the edible crab,Oziothelphusa senex senex.Rsc.Adv.,6(30):24959-24967.
Tada N,Nakao A,Hoshi H,Saka M,Kamata Y,2008.Vitellogenin,a biomarker for environmental estrogenic pollution,of Reeves'pond turtles:analysis of similarity for its amino acid sequence and cognate mRNA expression after exposure to estrogen.J.Vet.Med.Sci.,70(3):227-234.
Thornton JW,Need E,Crews D,2003.Resurrecting the ancestral steroid receptor:ancient origin of estrogen signaling.Science,301(5640):1714-1717.
Tominaga N,Ura K,Kawakami M,Kawaguchi T,Kohra S,Mitsui Y,Iguchi T,Arizono K,2003.Caenorhabditis elegans responses to specific steroid hormones.J.Heal.Sci.,49(1):28-33.
Tyler CR,vander Eerden B,Jobling S,Panter G,Sumpter JP,1996.Measurement of vitellogenin,a biomarker for exposure to oestrogenic chemicals,in a wide variety of cyprinid fish.J.Comp.Physiol.BBiochem.Sys.Environ.Physiol.,166(7):418-426.
Wienken CJ,Baaske P,Rothbauer U,Braun D,Duhr S,2010.Proteinbinding assays in biological liquids using microscale thermophoresis.Nat.Commun.,1(7):100.
Xu J,Wang YQ,Li ZQ,Ling L,Zeng BS,You L,Chen YZ,Aslam AF,Huang YP,Tan AJ,2014.Functional characterization of the vitellogenin promoter in the silkworm,Bombyx mori.Insect Mol.Biol.,23(5):550-557.
Yang C,Lin Y,Liu H,Shen G,Luo J,Zhang H,Peng Z,Chen E,Xing R,Han C,Xia Q,2014a.The Broad Complex isoform 2(Br C-Z2)transcriptional factor plays a critical role in vitellogenin transcription in the silkworm,Bombyx mori.Biochim.Biophys.Acta,1840(2014):2674-2684.
Yang CW,Lin Y,Shen GW,Chen EX,Wang YX,Luo J,Zhang HY,Xing RM,Xia QY,2014b.Female qualities in males:vitellogenin synthesis induced by ovary transplants into the male silkworm,Bombyx mori.Biochem.Biophys.Res.Commun.,453(1):31-36.
Yang HJ,Lee JW,Lee SH,Ryu JS,Kwak DH,Nam KS,Park YI,Lee YC,Jung KY,Choo YK,2010.Estrogenic activity produced by aqueous extracts of silkworm(Bombyx mori)pupae in ovariectomized rats.Am.J.Chin.Med.,38(1):89-97.
Yocum LB,Denlinger DL,Katlic NE,Brueggemeier RW,Mechoulam R,1987.A developmental profile of estrogen and androgen radioimmunoactive substances in the flesh fly,Sarcophaga crassipalpis.Insect Biochem.,17(8):1149-1153.
Zhao Y,Hu J,2012.Development of a molecular biomarker for detecting intersex after exposure of male medaka fish to synthetic estrogen.Environ.Toxicol.Chem.,31(8):1765-1773.
Zhao Y,Li Y,Lou G,Zhao L,Xu Z,Zhang Y,He F,2012.MiR-137targets estrogen-related receptor alpha and impairs the proliferative and migratory capacity of breast cancer cells.PLo S ONE,7(6):e39102.