BIOLOGY OF REPRODUCTION (2016) 94(3):60, 1–10 Published online before print 27 January 2016. DOI 10.1095/biolreprod.115.133330 Synthetic Steroid Hormones Regulated Cell Proliferation Through MicroRNA-34a-5p in Human Ovarian Endometrioma1 Chia-Yi Hsu,3 Tsung-Hua Hsieh,4 Cheng-Fang Tsai,3 Hung-Sheng Chen,4 Peir-In Liang,5 Ya-Ling Hsu,3 and Eing-Mei Tsai2,3,4,6 3Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City, Taiwan 4Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Downloaded from https://academic.oup.com/biolreprod/article/94/3/60, 1-10/2434415 by guest on 24 September 2021 Kaohsiung City, Taiwan 5Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung City, Taiwan 6Center for Research Resources and Development, Kaohsiung Medical University, Kaohsiung City, Taiwan ABSTRACT steroid hormones and suggest a potential mechanism for endometriosis treatment. Endometriosis is the hormone-dependent product of endo- metrial tissue found outside the uterus. Recently, micro-RNAs danazol, endometrial mesenchymal stem cells, medroxypro- (miRNAs) were shown to play a role in endometriotic lesion gesterone acetate, progesterone development. However, the mechanism of steroid hormones Downloaded from www.biolreprod.org. responsible for miRNA remains obscure. In the present study, INTRODUCTION we assayed for the effects of synthetic steroid hormones Endometriosis, defined as the presence of endometriallike (danazol, progesterone, and medroxyprogesterone acetate tissue outside the uterus, is a common estrogen-dependent [MPA]) on miRNAs in endometriosis. We used a global miRNA gynecological disorder with a complex, multifactorial etiology expression profile microarray to evaluate miRNA expression in that affects approximately 10%–15% of women of reproduc- endometrial mesenchymal stem cells (EN-MSCs) of ovarian tive age. Several theories explaining endometriosis pathogen- l endometrioma following treatment with 1 M danazol, esis have been proposed, including the widely accepted theory progesterone, or MPA. Furthermore, we selected candidate miRNAs whose expression changed more than fivefold and of retrograde transplantation and subsequent implantation of compared the effects of danazol, progesterone, and MPA endometrial tissue fragments into ectopic locations after treatments and also compared those results with controls in retrograde menstruation [1]. The theories of coelomic meta- EN-MSCs. Among those with a fivefold change, we found 13 plasia, lymphovascular metastasis, embryonic rest, and stem ectopically upregulated miRNAs in EN-MSCs. To understand cell involvement may also potentially explain the development the function of these 13 miRNAs, we subjected their sequences of endometriosis [2, 3]. However, endometriosis is an to Ingenuity Pathway Analysis. According to both the etiology enigmatic disease with unsolved pathogenesis. Recently, there and pathogenesis of endometriosis, we found that miR-199a- is growing evidence indicating that stem cells act as the root 5p and miR-34a-5p showed specific association with the etiology of endometriosis [2, 4–7]. This theory postulates that disease, including molecular and cellular functions. Steroid endometriotic lesions result from endometrial stem/progenitor hormone treatment elevated the levels of miR-199a-5p and cells [2]. The human endometrium is a dynamic tissue, miR-34a-5p. An inhibitor of miR-34a-5p also reduced the undergoing cycles of growth, differentiation, shedding, and synthetic steroid hormones effects on cell proliferation. In vivo regeneration, throughout a woman’s reproductive years. data revealed that miRNA levels in endometriotic lesions Therefore, endometrial stem/progenitor cells are thought to correlated with findings following in vitro synthetic hormone have regenerative capacity. Owing to these regenerative traits, treatment. Our data show the effects of synthetic steroid endometrial stem/progenitor cells may actually contribute to hormones on miRNA regulation. These findings contribute to endometriosis formation. In our previous study, we demon- our understanding of the molecular impact of the synthetic strated the presence of mesenchymal stem cells (MSCs) in stromal cells of ectopic endometrial tissues and established 1This work was supported by a grant from the Kaohsiung Medical MSCs from patients with endometriosis. The results of the University Hospital Research Fund (KMUH104-4R27 and KMUH103- study showed that ectopic endometrial MSCs (EN-MSCs) have 10V07), and Ministry of Science and Technology, Taiwan (Grant No. higher cell invasion ability in vitro and in vivo [8]. In addition, NSC102-2628-B-037-011-MY3 and 102-2632-B-037-001-MY3). 2 we also demonstrated that ectopic EN-MSCs application in the Correspondence: Eing-Mei Tsai, Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung nude mice can form the endometrioticlike lesion [9]. Therefore, Medical University, No. 100, Zihyou 1st Rd., Sanmin District, we used the ectopic EN-MSCs as the model for the study of Kaohsiung City 807, Taiwan. E-mail: [email protected] endometriosis and to investigate the effect of the synthetic steroid hormone. Received: 10 July 2015. In addition, endometriosis is a multifactorial disease caused First decision: 9 August 2015. by hormonal milieu [10], genetic predisposition [11, 12], and Accepted: 19 January 2016. environmental factors [13]. The high incidence of recurrence Ó 2016 by the Society for the Study of Reproduction, Inc. This article is following treatment illustrates that the established theories may available under a Creative Commons License 4.0 (Attribution-Non- not fully explain the etiology of endometriosis. Implanted Commercial), as described at http://creativecommons.org/licenses/by-nc/ 4.0 ectopic endometrial tissue can resist apoptosis [14, 15], invade eISSN: 1529-7268 http://www.biolreprod.org the extracellular matrix [16], induce neovascularization [17], ISSN: 0006-3363 and acquire steroidogenic capacity [18]. 1 Article 60 HSU ET AL. Steroid hormones are produced in the ovaries, and they adipogenic, osteogenic, and chondrogenic markers, FABP4, Runx2, and regulate the growth of endometrial tissue by stimulating or collagen II, respectively, were determined by quantitative RT-PCR (qPCR). inhibiting cell proliferation. Steroid hormones play a role in According to our previous reports [8], we collected the total population (which contains epithelium and stromal cells of ovarian endometrioma) after uterine diseases such as endometriosis. Disruption of steroid passing through 70 lm wire sieves. Then, passing through a 40 lm strainer hormone control of endometrial cell proliferation and differ- (which contains stromal cells), we purified MSCs from endometriotic stromal entiation can lead to endometriosis. Furthermore, implantation cells after limiting dilution and colony formation. We collected MSCs from the and maintenance of endometrial lesions are estrogen depen- large colony formation after identification. Those without large colony dent. formation are defined as non-MSCs. Eventually, three EN-MSC cultures were obtained by this process. EN-MSCs were cultured in Dulbecco modified Eagle Therefore, a variety of synthetic hormones, such as danazol, medium: Nutrient Mixture F12 (Gibco-Life Technologies) and MCDB 153 progesterone, and medroxyprogesterone acetate (MPA) have medium (Keratinocyte-SFM; Gibco-Life Technologies) (2:1, v/v) supplement- been contrived for the treatment of endometriosis [19–21]. ed with 10% fetal bovine serum (Gibco-Life Technologies), 2 mM N-acetyl-L- They have been widely used in clinical therapy. These drugs cysteine (Sigma-Aldrich), and 0.2 mM Asc-2P (Sigma-Aldrich). EN-MSCs act as a progestin or an androgenlike hormone inhibiting both were incubated at 378C under humidified atmosphere with 5% CO2. Downloaded from https://academic.oup.com/biolreprod/article/94/3/60, 1-10/2434415 by guest on 24 September 2021 ovarian estrogen production and the pituitary-based surge of luteinizing hormone. However, steroid-related molecular Chemicals and Reagents mechanisms—especially those related to micro-RNA (miRNA) Danazol, progesterone, MPA, and b-estradiol were purchased from Sigma- regulation in endometriosis—are not well known. Aldrich. Primers were commercially synthesized at Genemessenger Scientific The miRNAs are short (;19–22 nucleotides), highly Co. The primers used in this study were the following: 18S, 50-GAGGTAGT conserved, noncoding RNAs that control gene expression by GACGAAAAATAACAAT-30 (sense) and 50-TTGCCCTCCAATGGATCCT- 0 30 (antisense); NOTCH1, 50-GACCTCATCAACTCACACGC-30 (sense) and targeting a specific sequence within the 3 untranslated region 0 0 0 of mRNAs, resulting in mRNA degradation or repression of 5 -TATGATCCGTGATGTCCCGG-3 (antisense); b-ACTIN,5- GTGGGGCGCCCCAGGCACCA-30 (sense) and 50-CTCCTTAATGTCACG translation. The miRNAs have been implicated in the CACGATTTC-30 (antisense); CD29, 50-CTGCAAGAACGGGGTGAATG-30 regulation of cellular functions such as cell cycle progression (sense) and 50-CACAATGTCTACCAACACGCCC-30 (antisense); CD44, 50- Downloaded from www.biolreprod.org. [22], proliferation [22–25], apoptosis [26, 27], cell motility AGAAGGTGTGGGCAGAAGAA-30 (sense) and 50-AAATGCAC [28], and angiogenesis [9]. Therefore, aberrant miRNA CATTTCCTGAGA-30 (antisense); CD90, 5 0-CGCTCTCCTGCTAA CAGTCTT-30 (sense) and 50-CAGGCTGAACTCGTACTGGA-30 (antisense);