MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells

Minegishi T (2014) MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells. PLoS ONE 9(10): e108997. doi:10.1371/journal.pone.0108997 MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells Masayuki Iwamune 0 Kazuto Nakamura 0 Yoshikazu Kitahara 0 Takashi Minegishi 0 Meijia Zhang, China Agricultural University, China 0 1 Department of Obstetrics and Gynecology, Gunma University Graduate School of Medicine , Maebashi, Gunma , Japan , 2 Department of Gynecology, Gunma Prefectural Cancer Center , Oota, Gunma , Japan The 78-kilodalton glucose-regulated protein (GRP78) is a molecular chaperone that assists in protein assembly, folding, and translocation. Recently, our laboratory reported that GRP78 regulates the expression of luteinizing hormone-human chorionic gonadotropin receptor (LHR) in the early stage of corpus luteum formation. In this study, we investigated whether microRNAs (miRNAs), which post-transcriptionally regulate mRNA, are involved in the regulation mechanism of GRP78 in the ovary. A miRNA microarray was performed to analyze the overall miRNA expression profile, and the results indicated that 44 miRNAs were expressed highly after ovulation was induced. The results from a bio-informative database analysis and in vitro granulosa cell culture studies led us to focus on rno-miR-376a for further analysis. In both in vivo and in vitro studies, rnomiR-376a levels increased 12 h after human chorionic gonadotropin (hCG) administration. To elucidate whether rno-miR376a induced mRNA destabilization or translational repression of GRP78, rno-miR-376a was transfected into cultured granulosa cells, resulting in decreased GPR78 protein levels without an alteration in GRP78 mRNA levels. To confirm that rno-miR-376a binds to GRP78 mRNA, we cloned the 39-end of GRP78 mRNA (nucleotides 2439-2459) into a reporter vector that contained a Renilla luciferase coding region upstream of the cloning site. The luciferase assays revealed that rno-miR376a bound to the 39-end of GRP78 mRNA. From these data, we conclude that rno-miR-376a potentially negatively regulates GRP78 protein expression through translational repression at an early stage transition from the follicular phase to luteinization. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. In females, ovulationthe release of a mature and fertilizable oocyteis an essential process in the establishment of pregnancy. Follicle stimulating hormone (FSH) is a key factor that induces luteinizing hormone-human chorionic gonadotropin receptor (LHR) expression in the granulosa cells of pre-ovulatory follicles, whereby an LH surge triggers ovulation followed by corpus luteum formation [1,2,3]. Ovulation provokes a dramatic transformation of ovulated follicle to form the corpus luteum, which in turn induces synthesis of progesterone in order to sustain pregnancy. This indicates that granulosa cells are under stress, which has led to ovulation being considered an inflammation-like phenomenon [4,5]. When a cell is exposed to a variety of environmental and physiological stresses, the function of the endoplasmic reticulum (ER) is disturbed, and unfolded proteins accumulated in the ER [6]. In the ER, molecular chaperones are responsible for proper protein folding and for preventing aggregation of unfoldedintermediates, which could lead to cell death [7]. Recently, our laboratory determined that the 78-kilodalton glucose-regulated protein (GRP78), an ER-associated molecular chaperone that assists in proper protein folding to execute primary protein maturation in the ER [8,9], is involved in the recovery of LHR after its down-regulation [10]. Although GRP78 is known be an important molecule for the upregulation of LHR, the precise mechanism underlying the regulation of GRP78 expression in the ovary has not been fully elucidated. MicroRNAs (miRNAs) are non-coding RNAs (approximately 22 nucleotides) that regulate gene expression by binding to the 39untranslated regions of target mRNAs to induce the degradation of target mRNAs or to induce translational repression [11]. Within the past decade, miRNAs have been recognized as important regulators in many biological and cellular processes, such as cell proliferation, differentiation, apoptosis, and tumorigenesis [12,13,14]. Although the functions of miRNAs have not been elucidated fully, recent emerging evidence demonstrates that miRNAs are involved in ovarian follicular and luteal functions [15,16,17,18]. We have also reported that miR-136-3p targets LHR mRNA to induce the transient downregulation of LHR mRNA in granulosa cells after ovulation [19]. This result prompted us to search for miRNAs involved in the regulation of GRP78 expression. In the following experiments, we profiled the miRNAs that were expressed in PMSG-primed rat ovaries in which an injection of hCG induced ovulation. From that data and data obtained from a bio-informatics database, we focused on rno-miR-376a, which potentially binds to GRP78 mRNA, and characterized the function of rno-miR-376a in cultured granulosa cells. Materials and Methods Ethic Statement All procedures involving animals followed ethical principles according the NIH Guide for Care and Use of the Laboratory Animals and were approved by the Gunma University Animal Care and Use Committee (Permit Number: 12011). Hormones and reagents Recombinant FSH and purified hCG were supplied by Dr. A. Parlow and the National Hormone and Peptide Program (National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Torrance, CA, USA). Dulbecco modified Eagle medium (DMEM), DMEM/Hams Nutrient mixture F-12, diethylstilbestrol (DES) and b-Estradiol-Water Soluble were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Gentamicin sulfate and Fungizone were purchased from Invitrogen Corp. (Carlsbad, CA, USA). hCG for the in vivo study was provided by Schering-Plough Corp. (Osaka, Japan), and PMSG was provided by Asuka Pharmaceutical Co., Ltd. (Tokyo, Japan). Animals Female 21-day-old Wistar rats (Japan SLC, Inc., Hamamatsu, Japan) were housed in a temperature- and light-controlled room (12 h light, 12 h dark cycle; lights on at 0600 h) with food and water provided ad libitum. For the in vivo study, 21-day-old female rats received an injection of PMSG (30 IU, subcutaneous [sc]), followed by an injection of hCG (20 IU, sc) 48 h later. We then sacrificed the rats by decapitation to remove the ovaries at each time point after the injection of hCG. The ovaries were stored immediately in RNAlater Tissue Collection solution (Applied Biosystems, Foster City, CA, USA). F (...truncated)