MicroRNA-21 enhances estradiol production by inhibiting WT1 expression in granulosa cells

in Journal of Molecular Endocrinology
View More View Less
  • 1 Department of Animal Biosciences, University of Guelph, Guelph, Ontario, Canada
  • | 2 Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institute of Health, Bethesda, Maryland, USA

Correspondence should be addressed to J Li: jli@uoguelph.ca
Restricted access

In antral follicles, the transition of proliferative granulosa cells to estradiol-producing is critical for proper oocyte maturation. MicroRNAs are noncoding RNAs that play important roles in ovarian follicular development; however, this has yet to be fully characterized. MicroRNA-21 is significantly higher in granulosa cells isolated from large antral follicles compared to those from small antral follicles. To investigate the function of miR-21, porcine granulosa cells were transfected with miR-21 mimic or miR-21 targeted siRNA. Cells with the miR-21 mimic had higher aromatase expression and estradiol production but decreased WT1 expression. Conversely, cells with the miR-21 siRNA secreted less estradiol and had higher WT1 expression. We hypothesized that miR-21 promotes estradiol production by inhibiting WT1 protein synthesis. We found a potential miR-21 binding site in the 3’UTR of the WT1 transcript and performed a dual-luciferase reporter assay using the WT and mutated 3’UTR. Compared to the negative control, the miR-21 mimic induced a significant decrease in luciferase activity in the WT 3’UTR. This decrease was reversed when the 3’UTR was mutated, suggesting miR-21 targets this site to inhibit WT1 expression. We next transfected porcine granulosa cells with WT1 targeted siRNA and observed a significant increase in aromatase expression and estradiol secretion. We propose that miR-21 represses WT1 expression in granulosa cells to potentially promote aromatase expression and estradiol production. This study offers the first report of a microRNA regulating WT1 expression in granulosa cells and reveals the role of miR-21 in WT1’s regulation of estradiol production.

 

Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 615 615 615
Full Text Views 16 16 16
PDF Downloads 21 21 21
  • Adir M, Combelles CMH, Mansur A, Ophir L, Hourvitz A, Orvieto R, Dor J & Machtinger R 2017 Dibutyl phthalate impairs steroidogenesis and a subset of LH-dependent genes in cultured human mural granulosa cell in vitro. Reproductive Toxicology 69 1318. (https://doi.org/10.1016/j.reprotox.2016.12.007)

    • Search Google Scholar
    • Export Citation
  • Baley J & Li J 2012 MicroRNAs and ovarian function. Journal of Ovarian Research 5 1. (https://doi.org/10.1186/1757-2215-5-8)

  • Bhat-Nakshatri P, Wang G, Collins NR, Thomson MJ, Geistlinger TR, Carroll JS, Brown M, Hammond S, Srour EF & Liu Y et al.2009 Estradiol-regulated microRNAs control estradiol response in breast cancer cells. Nucleic Acids Research 37 48504861. (https://doi.org/10.1093/nar/gkp500)

    • Search Google Scholar
    • Export Citation
  • Britt KL, Saunders PK, McPherson SJ, Misso ML, Simpson ER & Findlay JK 2004 Estrogen actions on follicle formation and early follicle development. Biology of Reproduction 71 17121723. (https://doi.org/10.1095/biolreprod.104.028175)

    • Search Google Scholar
    • Export Citation
  • Carletti MZ, Fiedler SD & Christenson LK 2010 MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells. Biology of Reproduction 83 286295. (https://doi.org/10.1095/biolreprod.109.081448)

    • Search Google Scholar
    • Export Citation
  • Carthew RW & Sontheimer EJ 2009 Origins and mechanisms of miRNAs and siRNAs. Cell 136 642655. (https://doi.org/10.1016/j.cell.2009.01.035)

  • Casarini L & Crépieux P 2019 Molecular mechanisms of action of FSH. Frontiers in Endocrinology 10 305. (https://doi.org/10.3389/fendo.2019.00305)

    • Search Google Scholar
    • Export Citation
  • Chan JA, Krichevsky AM & Kosik KS 2005 MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Research 65 60296033. (https://doi.org/10.1158/0008-5472.CAN-05-0137)

    • Search Google Scholar
    • Export Citation
  • Chen J, Gao S, Wang C, Wang Z, Zhang H, Huang K, Zhou B, Li H, Yu Z & Wu et al.2016 Pathologically decreased expression of MIR-193a contributes to metastasis by targeting WT1-E-cadherin axis in non-small cell lung cancers. Journal of Experimental and Clinical Cancer Research 35 115. (https://doi.org/10.1186/s13046-016-0450-8)

    • Search Google Scholar
    • Export Citation
  • Chen M, Zhang L, Cui X, Lin X, Li Y, Wang Y, Wang Y, Qin Y, Chen D & Han C et al.2017 Wt1 directs the lineage specification of Sertoli and granulosa cells by repressing Sf1 expression. Development 144 4453. (https://doi.org/10.1242/dev.144105)

    • Search Google Scholar
    • Export Citation
  • Donadeu FX, Sontakke SD & Ioannidis J 2016 MicroRNA indicators of follicular steroidogenesis. Reproduction, Fertility, and Development 29 906912. (https://doi.org/10.1071/RD15282)

    • Search Google Scholar
    • Export Citation
  • Edson MA, Nagaraja AK & Matzuk MM 2009 The mammalian ovary from genesis to revelation. Endocrine Reviews 30 624712. (https://doi.org/10.1210/er.2009-0012)

    • Search Google Scholar
    • Export Citation
  • Fiedler SD, Carletti MZ, Hong X & Christenson LK 2008 Hormonal regulation of microRNA expression in periovulatory mouse mural granulosa cells. Biology of Reproduction 79 10301037. (https://doi.org/10.1095/biolreprod.108.069690)

    • Search Google Scholar
    • Export Citation
  • Fiorillo AA, Heier CR, Huang YF, Tully CB, Punga T & Punga AR 2020 Estrogen receptor, inflammatory, and FOXO transcription factors regulate expression of myasthenia gravis-associated circulating microRNAs. Frontiers in Immunology 11 151. (https://doi.org/10.3389/fimmu.2020.00151)

    • Search Google Scholar
    • Export Citation
  • Gao F, Zhang J, Wang X, Yang J, Chen D, Huff V & Liu YX 2014 Wt1 functions in ovarian follicle development by regulating granulosa cell differentiation. Human Molecular Genetics 23 333341. (https://doi.org/10.1093/hmg/ddt423)

    • Search Google Scholar
    • Export Citation
  • Gurates B, Amsterdam A, Tamura M, Yang S, Zhou J, Fang Z, Amin S, Sebastian S & Bulun SE 2003 WT1 and DAX-1 regulate SF-1-mediated human P450arom gene expression in gonadal cells. Molecular and Cellular Endocrinology 208 6175. (https://doi.org/10.1016/s0303-7207(0300198-9)

    • Search Google Scholar
    • Export Citation
  • Hsu SY, Kubo M, Chun SY, Haluska FG, Housman DE & Hsueh AJW 1995 Wilms’ tumor protein WT1 as an ovarian transcription factor: decreases in expression during follicle development and repression of inhibin-α gene promoter. Molecular Endocrinology 9 13561366. (https://doi.org/10.1210/mend.9.10.8544844)

    • Search Google Scholar
    • Export Citation
  • Hutvagner G & Zamore PD 2002 A microRNA in a multiple-turnover RNAi enzyme complex. Science 297 20562060. (https://doi.org/10.1126/science.1073827)

    • Search Google Scholar
    • Export Citation
  • Jamnongjit M & Hammes SR 2006 Ovarian steroids: the good, the bad, and the signals that raise them. Cell Cycle 5 11781183. (https://doi.org/10.4161/cc.5.11.2803)

    • Search Google Scholar
    • Export Citation
  • Jeppesen JV, Kristensen SG, Nielsen ME, Humaidan P, Dal Canto M, Fadini R, Schmidt KT, Ernst E & Andersen CY 2012 LH-receptor gene expression in human granulosa and cumulus cells from antral and preovulatory follicles. Journal of Clinical Endocrinology and Metabolism 97 15241531. (https://doi.org/10.1210/jc.2012-1427)

    • Search Google Scholar
    • Export Citation
  • Karakaya C, Guzeloglu-Kayisli O, Uyar A, Kallen AN, Babayev E, Bozkurt N, Unsal E, Karabacak O & Seli E 2015 Poor ovarian response in women undergoing in vitro fertilization is associated with altered microRNA expression in cumulus cells. Fertility and Sterility 103 1469 .e1147 6.e1. (https://doi.org/10.1016/j.fertnstert.2015.02.035)

    • Search Google Scholar
    • Export Citation
  • Kishi H, Kitahara Y, Imai F, Nakao K & Suwa H 2018 Expression of the gonadotropin receptors during follicular development. Reproductive Medicine and Biology 17 1119. (https://doi.org/10.1002/rmb2.12075)

    • Search Google Scholar
    • Export Citation
  • Krüger J & Rehmsmeier M 2006 RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Research 34 W451W454. (https://doi.org/10.1093/nar/gkl243)

    • Search Google Scholar
    • Export Citation
  • Kumar TR, Wang Y, Lu N & Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nature Genetics 15 201204. (https://doi.org/10.1038/ng0297-201)

    • Search Google Scholar
    • Export Citation
  • Lai EC 2002 Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation. Nature Genetics 30 363364. (https://doi.org/10.1038/ng865)

    • Search Google Scholar
    • Export Citation
  • Levallet J, Koskimies P, Rahman N & Huhtaniemi I 2001 The promoter of murine follicle-stimulating hormone receptor: functional characterization and regulation by transcription factor steroidogenic factor 1. Molecular Endocrinology 15 8092. (https://doi.org/10.1210/mend.15.1.0583)

    • Search Google Scholar
    • Export Citation
  • Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP & Burge CB 2003 Prediction of mammalian microRNA targets. Cell 115 787798. (https://doi.org/10.1016/s0092-8674(0301018-3)

    • Search Google Scholar
    • Export Citation
  • Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25 402408. (https://doi.org/10.1006/meth.2001.1262)

    • Search Google Scholar
    • Export Citation
  • Lyimo ZC, Nielen M, Ouweltjes W, Kruip TAM & van Eerdenburg FJCM 1999 Relationship among estradiol, cortisol and intensity of estrous behavior in dairy cattle. Theriogenology 53 17831795. (https://doi.org/10.1016/s0093-691x(0000314-9)

    • Search Google Scholar
    • Export Citation
  • Madsen CA, Perry GA, Mogck CL, Daly RF, MacNeil MD & Geary TW 2015 Effects of preovulatory estradiol on embryo survival and pregnancy establishment in beef cows. Animal Reproduction Science 158 96103. (https://doi.org/10.1016/j.anireprosci.2015.05.006)

    • Search Google Scholar
    • Export Citation
  • Makker A, Goel MM & Mahdi AA 2014 PI3K/PTEN/Akt and TSC/mTOR signaling pathways, ovarian dysfunction, and infertility: an update. Journal of Molecular Endocrinology 53 R103R118. (https://doi.org/10.1530/JME-14-0220)

    • Search Google Scholar
    • Export Citation
  • Makrigiannakis A, Amin K, Coukos G, Tilly JL & Coutifaris C 2000 Regulated expression and potential roles of p53 and Wilms’ tumor suppressor gene (WT1) during follicular development in the human ovary. Journal of Clinical Endocrinology and Metabolism 85 449459. (https://doi.org/10.1210/jcem.85.1.6246)

    • Search Google Scholar
    • Export Citation
  • Mani AM, Fenwick MA, Cheng Z, Sharma MK, Singh D & Wathes DC 2010 IGF1 induces up-regulation of steroidogenic and apoptotic regulatory genes via activation of phosphatidylinositol-dependent kinase/AKT in bovine granulosa cells. Reproduction 139 139151. (https://doi.org/10.1530/REP-09-0050)

    • Search Google Scholar
    • Export Citation
  • Mansur A, Adir M, Yerushalmi G, Hourvitz A, Gitman H, Yung Y, Orvieto R & Machtinger R 2016 Does BPA alter steroid hormone synthesis in human granulosa cells in vitro? Human Reproduction 31 15621569. (https://doi.org/10.1093/humrep/dew088)

    • Search Google Scholar
    • Export Citation
  • Mariscal DV, Bergfeld EGM, Cupp AS, Kojima FN, Fike KE, Sánchez T, Wehrman ME, Johnson RK, Kittok RJ & Ford JJ et al.1998 Concentrations of gonadotropins, estradiol and progesterone in sows selected on an index of ovulation rate and embryo survival. Animal Reproduction Science 54 3143. (https://doi.org/10.1016/s0378-4320(9800141-9)

    • Search Google Scholar
    • Export Citation
  • Mase Y, Ishibashi O, Ishikawa T, Takizawa T, Kiguchi K, Ohba T, Katabuchi H, Takeshita T & Takizawa T 2012 MiR-21 is enriched in the RNA-induced silencing complex and targets COL4A1 in human granulosa cell lines. Reproductive Sciences 19 10301040. (https://doi.org/10.1177/1933719112442245)

    • Search Google Scholar
    • Export Citation
  • Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST & Patel T 2007 MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 133 647658. (https://doi.org/10.1053/j.gastro.2007.05.022)

    • Search Google Scholar
    • Export Citation
  • Meng K, Wang X, He Y, Yang J, Wang H, Zhang Y & Quan F 2019 The Wilms tumor gene (WT1) (+/-KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cells. Biology of Reproduction 100 13441355. (https://doi.org/10.1093/biolre/ioz003)

    • Search Google Scholar
    • Export Citation
  • Michael MD, Kilgore MW, Morohashi K & Simpson ER 1995 Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. Journal of Biological Chemistry 270 1356113566. (https://doi.org/10.1074/jbc.270.22.13561)

    • Search Google Scholar
    • Export Citation
  • Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, Lim B & Rigoutsos I 2006 A pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes. Cell 126 12031217. (https://doi.org/10.1016/j.cell.2006.07.031)

    • Search Google Scholar
    • Export Citation
  • Murri M, Insenser M, Fernández-Durán E, San-Millán JL & Escobar-Morreale HF 2013 Effects of polycystic ovary syndrome (PCOS), sex hormones, and obesity on circulating miRNA-21, miRNA-27b, miRNA-103, and miRNA-155 expression. Journal of Clinical Endocrinology and Metabolism 98 E1835E1844. (https://doi.org/10.1210/jc.2013-2218)

    • Search Google Scholar
    • Export Citation
  • Nachtigal MW, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN, Hammer GD & Ingraham HA 1998 Wilms’ tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression. Cell 93 445454. (https://doi.org/10.1016/s0092-8674(0081172-1)

    • Search Google Scholar
    • Export Citation
  • Naji M, Aleyasin A, Nekoonam S, Arefian E, Mahdian R & Amidi F 2017 Differential expression of miR-93 and miR-21 in granulosa cells and follicular fluid of polycystic ovary syndrome associating with different phenotypes. Scientific Reports 7 14671. (https://doi.org/10.1038/s41598-017-13250-1)

    • Search Google Scholar
    • Export Citation
  • Orisaka M, Mizutani T, Tajima K, Orisaka S, Shukunami KI, Miyamoto K & Kotsuji F 2006 Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle. Molecular Reproduction and Development 73 737744. (https://doi.org/10.1002/mrd.20246)

    • Search Google Scholar
    • Export Citation
  • Pan B & Li J 2018 MicroRNA-21 up-regulates metalloprotease by down-regulating TIMP3 during cumulus cell-oocyte complex in vitro maturation. Molecular and Cellular Endocrinology 477 2938. (https://doi.org/10.1016/j.mce.2018.05.011)

    • Search Google Scholar
    • Export Citation
  • Pawlak P, Warzych E, Cieslak A, Malyszka N, Maciejewska E, Madeja ZE & Lechniak D 2018 The consequences of porcine IVM medium supplementation with follicular fluid become reflected in embryo quality, yield and gene expression patterns. Scientific Reports 8 15306. (https://doi.org/10.1038/s41598-018-33550-4)

    • Search Google Scholar
    • Export Citation
  • Petersen SL, Ottem EN & Carpenter CD 2003 Direct and indirect regulation of gonadotropin-releasing hormone neurons by estradiol. Biology of Reproduction 69 17711778. (https://doi.org/10.1095/biolreprod.103.019745)

    • Search Google Scholar
    • Export Citation
  • Regan SLP, Knight PG, Yovich JL, Leung Y, Arfuso F & Dharmarajan A 2018 Granulosa cell apoptosis in the ovarian follicle – a changing view. Frontiers in Endocrinology 9 61. (https://doi.org/10.3389/fendo.2018.00061)

    • Search Google Scholar
    • Export Citation
  • Reizner N, Maor S, Sarfstein R, Abramovitch S, Welshons WV, Curran EM, Lee AV & Werner H 2005 The WT1 Wilms’ tumor suppressor gene product interacts with estrogen receptor-α and regulates IGF-I receptor gene transcription in breast cancer cells. Journal of Molecular Endocrinology 35 135144. (https://doi.org/10.1677/jme.1.01761)

    • Search Google Scholar
    • Export Citation
  • Reza AMMT, Choi YJ, Han SG, Song H, Park C, Hong K & Kim JH 2019 Roles of microRNAs in mammalian reproduction: from the commitment of germ cells to peri-implantation embryos. Biological Reviews of the Cambridge Philosophical Society 94 415438. (https://doi.org/10.1111/brv.12459)

    • Search Google Scholar
    • Export Citation
  • Robker RL & Richards JS 1998 Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27(kip1) Molecular Endocrinology 12 924940. (https://doi.org/10.1210/mend.12.7.0138)

    • Search Google Scholar
    • Export Citation
  • Roh J, Bae J, Lee K, Mayo K, Shea L & Woodruff TK 2009 Regulation of Wilms’ tumor gene expression by nerve growth factor and follicle-stimulating hormone in the immature mouse ovary. Fertility and Sterility 91 (Supplement) 14511454. (https://doi.org/10.1016/j.fertnstert.2008.07.012)

    • Search Google Scholar
    • Export Citation
  • Ruan Q, Wang P, Wang T, Qi J, Wei M, Wang S, Fan T, Johnson D, Wan X & Shi W et al.2014 MicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2. Cell Death and Disease 5 e1095. (https://doi.org/10.1038/cddis.2014.47)

    • Search Google Scholar
    • Export Citation
  • Ryan BC, Werner TS, Howard PL & Chow RL 2016 ImiRP: a computational approach to microRNA target site mutation. BMC Bioinformatics 17 190. (https://doi.org/10.1186/s12859-016-1057-y)

    • Search Google Scholar
    • Export Citation
  • Sabry R, Saleh AC, Stalker L, LaMarre J & Favetta LA 2021 Effect of bisphenol A and bisphenol S on microRNA expression during in bovine (Bos taurus) oocyte maturation and early embryo development. Reproductive Toxicology 99 96108. (https://doi.org/10.1016/j.reprotox.2020.12.001)

    • Search Google Scholar
    • Export Citation
  • Sacchi S, D’Ippolito G, Sena P, Marsella T, Tagliasacchi D, Maggi E, Argento C, Tirelli A, Giulini S & La Marca A 2016 The anti-Müllerian hormone (AMH) acts as a gatekeeper of ovarian steroidogenesis inhibiting the granulosa cell response to both FSH and LH. Journal of Assisted Reproduction and Genetics 33 95100. (https://doi.org/10.1007/s10815-015-0615-y)

    • Search Google Scholar
    • Export Citation
  • Si ML, Zhu S, Wu H, Lu Z, Wu F & Mo YY 2007 miR-21-mediated tumor growth. Oncogene 26 27992803. (https://doi.org/10.1038/sj.onc.1210083)

  • Sontakke SD, Mohammed BT, McNeilly AS & Donadeu FX 2014 Characterization of microRNAs differentially expressed during bovine follicle development. Reproduction 148 271283. (https://doi.org/10.1530/REP-14-0140)

    • Search Google Scholar
    • Export Citation
  • Stocco C 2008 Aromatase expression in the ovary: hormonal and molecular regulation. Steroids 73 473487. (https://doi.org/10.1016/j.steroids.2008.01.017)

    • Search Google Scholar
    • Export Citation
  • Toms D, Pan B & Li J 2018 Endocrine regulation in the ovary by microRNA during the estrous cycle. Frontiers in Endocrinology 8 19. (https://doi.org/10.3389/fendo.2017.00378)

    • Search Google Scholar
    • Export Citation
  • Tscherner A, Brown AC, Stalker L, Kao J, Dufort I, Sirard MA & LaMarre J 2018 STAT3 signaling stimulates miR-21 expression in bovine cumulus cells during in vitro oocyte maturation. Scientific Reports 8 11527. (https://doi.org/10.1038/s41598-018-29874-w)

    • Search Google Scholar
    • Export Citation
  • Velthut-Meikas A, Simm J, Tuuri T, Tapanainen JS, Metsis M & Salumets A 2013 Research resource: small RNA-seq of human granulosa cells reveals miRNAs in FSHR and aromatase genes. Molecular Endocrinology 27 11281141. (https://doi.org/10.1210/me.2013-1058)

    • Search Google Scholar
    • Export Citation
  • Wang XL, Wu Y, Tan LB, Tian Z, Liu JH, Zhu DS & Zeng SM 2012 Follicle-stimulating hormone regulates pro-apoptotic protein Bcl-2-interacting mediator of cell death-extra long (Bim EL)-induced porcine granulosa cell apoptosis. Journal of Biological Chemistry 287 1016610177. (https://doi.org/10.1074/jbc.M111.293274)

    • Search Google Scholar
    • Export Citation
  • Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y & Klinge CM 2009 Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids Research 37 25842595. (https://doi.org/10.1093/nar/gkp117)

    • Search Google Scholar
    • Export Citation
  • Yang S, Zhang Y, Zhao X, Wang J & Shang J 2016 MicroRNA-361 targets Wilms’ tumor 1 to inhibit the growth, migration and invasion of non-small-cell lung cancer cells. Molecular Medicine Reports 14 54155421. (https://doi.org/10.3892/mmr.2016.5858)

    • Search Google Scholar
    • Export Citation
  • Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S & Madden TL 2012 Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13 134. (https://doi.org/10.1186/1471-2105-13-134)

    • Search Google Scholar
    • Export Citation
  • Yoon O & Roh J 2012 Regulation of FSH receptor expression by the Wilms’ tumor 1 gene product (WT1) in immature rat granulosa cells. Molecular Reproduction and Development 79 368368. (https://doi.org/10.1002/mrd.22036)

    • Search Google Scholar
    • Export Citation