| Summary: | 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.
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