title: MicroRNA-376a regulates 78-kilodalton glucose-regulated protein expression in rat granulosa cells.
abstract: 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, rno-miR-376a levels increased 12 h after human chorionic gonadotropin (hCG) administration. To elucidate whether rno-miR-376a 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 3'-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-miR-376a bound to the 3'-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.
authors.affiliation: Department of Obstetrics and Gynecology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Iwamune
authors.affiliation: Department of Gynecology, Gunma Prefectural Cancer Center, Oota, Gunma, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Nakamura
authors.affiliation: Department of Obstetrics and Gynecology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Kitahara
authors.affiliation: Department of Obstetrics and Gynecology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Minegishi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D034461:Luteinization / Q000502:physiology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D010053:Ovary / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D006063:Chorionic Gonadotropin; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D035683:MicroRNAs