绵羊miR-150启动子的鉴定及生殖激素对其转录调控研究
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摘要
旨在对绵羊miR-150基因的启动子进行鉴定以及探索生殖激素对miR-150启动子转录调控的影响。本研究利用生物学软件预测雌性绵羊(小尾寒羊)卵巢miR-150启动子区域及繁殖相关转录因子结合位点,构建重组质粒,并利用双荧光素酶报告基因检测miR-150的启动子活性。通过在无血清培养基内添加FSH、LH和E_2,进行miR-150启动子重组质粒转染并检测miR-150启动子活性。结果,分别利用Promoter 2.0和PROMO软件预测出miR-150启动子区域和转录起始位点,并获得了与繁殖相关转录因子结合位点。所预测的绵羊miR-150启动子片段序列长度为582 bp,且成功构建miR-150启动子质粒,并确定绵羊颗粒细胞的最佳转染效率(脂质体∶质粒DNA=1∶1)和质粒最佳转染比例(50∶1),从而验证了所预测的区域具有启动子活性。此外,FSH、LH和E_2处理组与对照组(无激素组)相比,miR-150启动子的活性均显著下调(P<0.05)。结果表明,生殖激素可以通过影响绵羊miR-150启动子活性来调控miR-150基因的表达,为miR-150调控卵泡发育的机制研究提供了新思路。
The aim of this study was to identify the promoter of miR-150 gene and explore the effect of reproductive hormone on its transcription regulation in sheep. The miR-150 promoter region of ovary in female sheep(Small-tail Han sheep) and the reproduction-related transcription factor binding site were predicted by biological softwares, then recombinant plasmids were constructed and the dual luciferase reporter gene was used to detect the activity of miR-150 promoter. Moreover, the miR-150 promoter recombinant plasmid was transfected and miR-150 promoter activity was detected by adding FSH, LH and E_2 in serum-free medium. The miR-150 promoter region and transcription initiation site were respectively predicted using Promoter 2.0 and PROMO, and transcription factor binding sites associated with reproduction were obtained. The predicted length of the sheep miR-150 promoter fragment was 582 bp, and the miR-150 promoter plasmid was successfully constructed. The optimal transfection efficiency(liposomes∶plasmid DNA=1∶1) of the sheep granulosa cells and the optimal transfection proportion(50∶1) of the plasmid were determined. Thus, the predicted region with significant promoter activity was verified. In addition, the activity of the miR-150 promoter was significantly down-regulated in the FSH, LH and E_2 treatment groups compared with the control group(hormone-free group)(P<0.05). The results indicated that reproductive hormone could regulate the expression of miR-150 by affecting the miR-150 promoter activity, which provided a new idea for studying the mechanism of miR-150 regulating ovine follicular development.
The aim of this study was to identify the promoter of miR-150 gene and explore the effect of reproductive hormone on its transcription regulation in sheep. The miR-150 promoter region of ovary in female sheep(Small-tail Han sheep) and the reproduction-related transcription factor binding site were predicted by biological softwares, then recombinant plasmids were constructed and the dual luciferase reporter gene was used to detect the activity of miR-150 promoter. Moreover, the miR-150 promoter recombinant plasmid was transfected and miR-150 promoter activity was detected by adding FSH, LH and E_2 in serum-free medium. The miR-150 promoter region and transcription initiation site were respectively predicted using Promoter 2.0 and PROMO, and transcription factor binding sites associated with reproduction were obtained. The predicted length of the sheep miR-150 promoter fragment was 582 bp, and the miR-150 promoter plasmid was successfully constructed. The optimal transfection efficiency(liposomes∶plasmid DNA=1∶1) of the sheep granulosa cells and the optimal transfection proportion(50∶1) of the plasmid were determined. Thus, the predicted region with significant promoter activity was verified. In addition, the activity of the miR-150 promoter was significantly down-regulated in the FSH, LH and E_2 treatment groups compared with the control group(hormone-free group)(P<0.05). The results indicated that reproductive hormone could regulate the expression of miR-150 by affecting the miR-150 promoter activity, which provided a new idea for studying the mechanism of miR-150 regulating ovine follicular development.
引文
[1] 李琴.microRNA对哺乳动物卵泡发育的影响[J].畜牧兽医学报,2018,49(12):2558-2566.LI Q.Roles of microRNA on mammalian follicle development[J].Acta Veterinaria et Zootechnica Sinica,2018,49(12):2558-2566.(in Chinese)
[2] BARTEL D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[3] YIN M M,Lü M R,YAO G D,et al.Transactivation of microRNA-383 by steroidogenic factor-1 promotes estradiol release from mouse ovarian granulosa cells by targeting RBMS1[J].Mol Endocrinol,2012,26(7):1129-1143.
[4] YAO G D,YIN M M,LIAN J,et al.MicroRNA-224 is involved in transforming growth factor-β-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4[J].Mol Endocrinol,2010,24(3):540-551.
[5] CAYO-COLCA I S,YAMAGAMI Y,PHAN T C,et al.A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles[J].Theriogenology,2011,75(9):1602-1612.
[6] ZAMAH A M,HSIEH M,CHEN J,et al.Human oocyte maturation is dependent on LH-stimulated accumulation of the epidermal growth factor-like growth factor,amphiregulin[J].Hum Reprod,2010,25(10):2569-2578.
[7] YAO N,YANG B Q,LIU Y,et al.Follicle-stimulating hormone regulation of microRNA expression on progesterone production in cultured rat granulosa cells[J].Endocrine,2010,38(2):158-166.
[8] 郭文艳,丛晶,吴效科.类固醇激素在卵泡生长发育过程中的作用[J].世界中西医结合杂志,2008,3(2):120-122.GUO W Y,CONG J,WU X K.The effect of steroid hormones in the growth and development of follicles[J].World Journal of Integrated Traditional and Western Medicine,2008,3(2):120-122.(in Chinese)
[9] KLINGE C M.Estrogen regulation of microRNA expression[J].Curr Genomics,2009,10(3):169-183.
[10] SELIMOGLU-BUET D,RIVIèRE J,GHAMLOUCH H,et al.A miR-150/TET3 pathway regulates the generation of mouse and human non-classical monocyte subset[J].Nat Commun,2018,9(1):5455.
[11] CHEN X X,XU X N,PAN B,et al.miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer[J].Aging (Albany NY),2018,10(11):3421-3437.
[12] SRUTOVA K,CURIK N,BURDA P,et al.BCR-ABL1 mediated miR-150 downregulation through MYC contributed to myeloid differentiation block and drug resistance in chronic myeloid leukemia[J].Haematologica,2018,103(12):2016-2025,doi:10.3324/ haematol.2018.193086.
[13] KHAN H A,ZHAO Y,WANG L,et al.Identification of miRNAs during mouse postnatal ovarian development and superovulation[J].J Ovarian Res,2015,8:44.
[14] 郑全辉,田枫,李文婷,等.miR-150基因缺失对小鼠繁殖和血液学指标的影响[J].中国免疫学杂志,2016,32(10):1409-1412.ZHENG Q H,TIAN F,LI W T,et al.miR-150 deletion affects breeding and hematologic parameters of mice[J].Chinese Journal of Immunology,2016,32(10):1409-1412.(in Chinese)
[15] 苗艳平,陈烨,王涵,等.吐鲁番黑羊黄体期和卵泡期卵巢microRNA差异表达分析[J].农业生物技术学报,2018,26(4):660-669.MIAO Y P,CHEN Y,WANG H,et al.Analysis of differentially expressed microRNA in luteal and follicular ovaries of Turpan Black Sheep (Ovis aries)[J].Journal of Agricultural Biotechnology,2018,26(4):660-669.(in Chinese)
[16] 苗艳平,刘若男,魏彦辉,等.绵羊卵巢oar-mir-150靶向调节类固醇激素合成急性调节蛋白基因的表达[J].农业生物技术学报,2018,26(2):234-245.MIAO Y P,LIU R N,WEI Y H,et al.Target regulation of the expression of steroidogenicacute regulatory protein gene in sheep (Ovis aries) ovaries by oar-mir-150[J].Journal of Agricultural Biotechnology,2018,26(2):234-245.(in Chinese)
[17] ZHOU R Y,MIAO Y P,LI Y M,et al.MicroRNA-150 promote apoptosis of ovine ovarian granulosa cells by targeting STAR gene[J].Theriogenology,2019,127:66-71.
[18] DE ALMEIDA CHUFFA ■,et al.The role of sex hormones and steroid receptors on female reproductive cancers[J].Steroids,2017,118:93-108.
[19] UDHANE S S,PANDEY A V,HOFER G,et al.Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis[J].Sci Rep,2015,5:10132.
[20] KRIKUN G,LOCKWOOD C J.Steroid hormones,endometrial gene regulation and the Sp1 family of proteins[J].J Soc Gynecol Investig,2002,9(6):329-334.
[21] LAURSEN K B,GUDAS L J.Combinatorial knockout of RARα,RARβ,and RARγ completely abrogates transcriptional responses to retinoic acid in murine embryonic stem cells[J].J Biol Chem,2018,293(30):11891-11900.
[22] BORGES L,IACOVINO M,MAYERHOFER T,et al.A critical role for endoglin in the emergence of blood during embryonic development[J].Blood,2012,119(23):5417-5428.
[23] 陈国珍,朱静,田杰.组蛋白乙酰化酶p300和CREB结合蛋白在小鼠胚胎心发育中的时序表达[J].解剖学杂志,2008,31(4):480-482.CHEN G Z,ZHU J,TIAN J.Spatiotemporal expressions of histone acetyltransferases p300 and CREB binding protein during mouse embryonic heart development[J].Chinese Journal of Anatomy,2008,31(4):480-482.(in Chinese)
[24] 蒋静.影响小鼠胚胎时期心脏发育的相关蛋白[J].实验与检验医学,2014,32(2):153-156,171.JIANG J.Related proteins of affecting mouse embryonic heart development[J].Experimental and Laboratory Medicine,2014,32(2):153-156,171.(in Chinese)
[25] EDQVIST P H,MYERS S M,HALLB??K F.Early identification of retinal subtypes in the developing,pre-laminated chick retina using the transcription factors Prox1,Lim1,Ap2α,Pax6,Isl1,Isl2,Lim3 and Chx10[J].Eur J Histochem,2006,50(2):147-154.
[26] PHELPS D E,DRESSLER G R.Identification of novel Pax-2 binding sites by chromatin precipitation[J].J Biol Chem,1996,271(14):7978-7985.
[27] DUPREY P,CHOWDHURY K,DRESSLER G R,et al.A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine[J].Genes Dev,1988,2(12A):1647-1654.
[28] 常迪,董晓晨,郭勇,等.不同培养方法及培养密度对绵羊腔前卵泡发育的影响[J].中国农学通报,2017,33(8):130-134.CHANG D,DONG X C,GUO Y,et al.Effect of different culture methods and densities on sheep preantral follicles development[J].Chinese Agricultural Science Bulletin,2017,33(8):130-134.(in Chinese)
[29] WU J,XU B,WANG W.Effects of luteinizing hormone and follicle stimulating hormone on the developmental competence of porcine preantral follicle oocytes grown in vitro[J].J Assist Reprod Genet,2007,24(9):419-424.
[30] QUIRK S M,COWAN R G,HARMAN R M.The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle[J].J Endocrinol,2006,189(3):441-453.
[31] YAVAS Y,JOHNSON W H,WALTON J S.Modification of follicular dynamics by exogenous FSH and progesterone,and the induction of ovulation using hCG in postpartum beef cows[J].Theriogenology,1999,52(6):949-963.
[32] 杨世华.几种生殖激素对体外培养的牦牛卵泡颗粒细胞凋亡的影响[D].兰州:甘肃农业大学,2002.YANG S H.Effects of several reproductive hormones on apoptosis of ovarian granulose cells of Yaks in vitro[J].Lanzhou:Gansu Agricultural University,2002.(in Chinese)
[33] SARAIVA M V A,CELESTINO J J H,CHAVES R N,et al.Influence of different concentrations of LH and FSH on in vitro caprine primordial ovarian follicle development[J].Small Ruminant Res,2008,78(1-3):87-95.
[34] 孙健红,张涌,郑月茂,等.ERα免疫反应产物在山羊卵巢中的分布[J].畜牧兽医学报,2006,37(9):920-923.SUN J H,ZHANG Y,ZHENG Y M,et al.Distribution of estrogen receptor α immunoreactivity in goats ovary[J].Acta Veterinaria et Zootechnica Sinica,2006,37(9):920-923.(in Chinese)
[35] ORTEGA I,GARCíA-VELASCO J A,PELLICER A.Ovarian manipulation in ART:going beyond physiological standards to provide best clinical outcomes[J].J Assist Reprod Genet,2018,35(10):1751-1762.
[2] BARTEL D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[3] YIN M M,Lü M R,YAO G D,et al.Transactivation of microRNA-383 by steroidogenic factor-1 promotes estradiol release from mouse ovarian granulosa cells by targeting RBMS1[J].Mol Endocrinol,2012,26(7):1129-1143.
[4] YAO G D,YIN M M,LIAN J,et al.MicroRNA-224 is involved in transforming growth factor-β-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4[J].Mol Endocrinol,2010,24(3):540-551.
[5] CAYO-COLCA I S,YAMAGAMI Y,PHAN T C,et al.A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles[J].Theriogenology,2011,75(9):1602-1612.
[6] ZAMAH A M,HSIEH M,CHEN J,et al.Human oocyte maturation is dependent on LH-stimulated accumulation of the epidermal growth factor-like growth factor,amphiregulin[J].Hum Reprod,2010,25(10):2569-2578.
[7] YAO N,YANG B Q,LIU Y,et al.Follicle-stimulating hormone regulation of microRNA expression on progesterone production in cultured rat granulosa cells[J].Endocrine,2010,38(2):158-166.
[8] 郭文艳,丛晶,吴效科.类固醇激素在卵泡生长发育过程中的作用[J].世界中西医结合杂志,2008,3(2):120-122.GUO W Y,CONG J,WU X K.The effect of steroid hormones in the growth and development of follicles[J].World Journal of Integrated Traditional and Western Medicine,2008,3(2):120-122.(in Chinese)
[9] KLINGE C M.Estrogen regulation of microRNA expression[J].Curr Genomics,2009,10(3):169-183.
[10] SELIMOGLU-BUET D,RIVIèRE J,GHAMLOUCH H,et al.A miR-150/TET3 pathway regulates the generation of mouse and human non-classical monocyte subset[J].Nat Commun,2018,9(1):5455.
[11] CHEN X X,XU X N,PAN B,et al.miR-150-5p suppresses tumor progression by targeting VEGFA in colorectal cancer[J].Aging (Albany NY),2018,10(11):3421-3437.
[12] SRUTOVA K,CURIK N,BURDA P,et al.BCR-ABL1 mediated miR-150 downregulation through MYC contributed to myeloid differentiation block and drug resistance in chronic myeloid leukemia[J].Haematologica,2018,103(12):2016-2025,doi:10.3324/ haematol.2018.193086.
[13] KHAN H A,ZHAO Y,WANG L,et al.Identification of miRNAs during mouse postnatal ovarian development and superovulation[J].J Ovarian Res,2015,8:44.
[14] 郑全辉,田枫,李文婷,等.miR-150基因缺失对小鼠繁殖和血液学指标的影响[J].中国免疫学杂志,2016,32(10):1409-1412.ZHENG Q H,TIAN F,LI W T,et al.miR-150 deletion affects breeding and hematologic parameters of mice[J].Chinese Journal of Immunology,2016,32(10):1409-1412.(in Chinese)
[15] 苗艳平,陈烨,王涵,等.吐鲁番黑羊黄体期和卵泡期卵巢microRNA差异表达分析[J].农业生物技术学报,2018,26(4):660-669.MIAO Y P,CHEN Y,WANG H,et al.Analysis of differentially expressed microRNA in luteal and follicular ovaries of Turpan Black Sheep (Ovis aries)[J].Journal of Agricultural Biotechnology,2018,26(4):660-669.(in Chinese)
[16] 苗艳平,刘若男,魏彦辉,等.绵羊卵巢oar-mir-150靶向调节类固醇激素合成急性调节蛋白基因的表达[J].农业生物技术学报,2018,26(2):234-245.MIAO Y P,LIU R N,WEI Y H,et al.Target regulation of the expression of steroidogenicacute regulatory protein gene in sheep (Ovis aries) ovaries by oar-mir-150[J].Journal of Agricultural Biotechnology,2018,26(2):234-245.(in Chinese)
[17] ZHOU R Y,MIAO Y P,LI Y M,et al.MicroRNA-150 promote apoptosis of ovine ovarian granulosa cells by targeting STAR gene[J].Theriogenology,2019,127:66-71.
[18] DE ALMEIDA CHUFFA ■,et al.The role of sex hormones and steroid receptors on female reproductive cancers[J].Steroids,2017,118:93-108.
[19] UDHANE S S,PANDEY A V,HOFER G,et al.Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis[J].Sci Rep,2015,5:10132.
[20] KRIKUN G,LOCKWOOD C J.Steroid hormones,endometrial gene regulation and the Sp1 family of proteins[J].J Soc Gynecol Investig,2002,9(6):329-334.
[21] LAURSEN K B,GUDAS L J.Combinatorial knockout of RARα,RARβ,and RARγ completely abrogates transcriptional responses to retinoic acid in murine embryonic stem cells[J].J Biol Chem,2018,293(30):11891-11900.
[22] BORGES L,IACOVINO M,MAYERHOFER T,et al.A critical role for endoglin in the emergence of blood during embryonic development[J].Blood,2012,119(23):5417-5428.
[23] 陈国珍,朱静,田杰.组蛋白乙酰化酶p300和CREB结合蛋白在小鼠胚胎心发育中的时序表达[J].解剖学杂志,2008,31(4):480-482.CHEN G Z,ZHU J,TIAN J.Spatiotemporal expressions of histone acetyltransferases p300 and CREB binding protein during mouse embryonic heart development[J].Chinese Journal of Anatomy,2008,31(4):480-482.(in Chinese)
[24] 蒋静.影响小鼠胚胎时期心脏发育的相关蛋白[J].实验与检验医学,2014,32(2):153-156,171.JIANG J.Related proteins of affecting mouse embryonic heart development[J].Experimental and Laboratory Medicine,2014,32(2):153-156,171.(in Chinese)
[25] EDQVIST P H,MYERS S M,HALLB??K F.Early identification of retinal subtypes in the developing,pre-laminated chick retina using the transcription factors Prox1,Lim1,Ap2α,Pax6,Isl1,Isl2,Lim3 and Chx10[J].Eur J Histochem,2006,50(2):147-154.
[26] PHELPS D E,DRESSLER G R.Identification of novel Pax-2 binding sites by chromatin precipitation[J].J Biol Chem,1996,271(14):7978-7985.
[27] DUPREY P,CHOWDHURY K,DRESSLER G R,et al.A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine[J].Genes Dev,1988,2(12A):1647-1654.
[28] 常迪,董晓晨,郭勇,等.不同培养方法及培养密度对绵羊腔前卵泡发育的影响[J].中国农学通报,2017,33(8):130-134.CHANG D,DONG X C,GUO Y,et al.Effect of different culture methods and densities on sheep preantral follicles development[J].Chinese Agricultural Science Bulletin,2017,33(8):130-134.(in Chinese)
[29] WU J,XU B,WANG W.Effects of luteinizing hormone and follicle stimulating hormone on the developmental competence of porcine preantral follicle oocytes grown in vitro[J].J Assist Reprod Genet,2007,24(9):419-424.
[30] QUIRK S M,COWAN R G,HARMAN R M.The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle[J].J Endocrinol,2006,189(3):441-453.
[31] YAVAS Y,JOHNSON W H,WALTON J S.Modification of follicular dynamics by exogenous FSH and progesterone,and the induction of ovulation using hCG in postpartum beef cows[J].Theriogenology,1999,52(6):949-963.
[32] 杨世华.几种生殖激素对体外培养的牦牛卵泡颗粒细胞凋亡的影响[D].兰州:甘肃农业大学,2002.YANG S H.Effects of several reproductive hormones on apoptosis of ovarian granulose cells of Yaks in vitro[J].Lanzhou:Gansu Agricultural University,2002.(in Chinese)
[33] SARAIVA M V A,CELESTINO J J H,CHAVES R N,et al.Influence of different concentrations of LH and FSH on in vitro caprine primordial ovarian follicle development[J].Small Ruminant Res,2008,78(1-3):87-95.
[34] 孙健红,张涌,郑月茂,等.ERα免疫反应产物在山羊卵巢中的分布[J].畜牧兽医学报,2006,37(9):920-923.SUN J H,ZHANG Y,ZHENG Y M,et al.Distribution of estrogen receptor α immunoreactivity in goats ovary[J].Acta Veterinaria et Zootechnica Sinica,2006,37(9):920-923.(in Chinese)
[35] ORTEGA I,GARCíA-VELASCO J A,PELLICER A.Ovarian manipulation in ART:going beyond physiological standards to provide best clinical outcomes[J].J Assist Reprod Genet,2018,35(10):1751-1762.
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