Role of microRNA-136-3p on the Expression of Luteinizing Hormone-Human Chorionic Gonadotropin Receptor mRNA in Rat Ovaries1

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that interact with mRNAs and trigger either translation repression or RNA cleavage of target genes. In this study, we investigated whether miRNA was involved in down-regulation of the luteinizing hormone receptor (LHR) in rat ovaries. An miRNA microarray was used to analyze the overall miRNA expression profile of rat ovaries in association with the down-regulation of LHR mRNA. We found that 23 miRNAs were highly expressed during this period. Combining these results with data from a bioinformatics database, clustering analysis led us to focus on miR-136-3p for further analysis. In both in vivo and in vitro studies, miR-136-3p expression levels were increased at 6 h after human chorionic gonadotropin (hCG) administration, concurrent with downregulation of LHR mRNA. Moreover, transfection of cultured granulosa cells with miR-136-3p resulted in a significant decrease in LHR mRNA levels in comparison with those of cells transfected with negative control. In contrast, transfection with a miR-136-3p inhibitor increased LHR mRNA levels. Finally, cotransfection of granulosa cells with a miR-136-3p inhibitor and a reporter vector containing the 3 0 -untranslated region (UTR) of LHR mRNA and Renilla luciferase coding sequence revealed that miR-136-3p bound directly to the 3 0 -UTR of LHR mRNA. These data demonstrated that miR-136-3p participated in the down-regulation of LHR mRNA by binding directly to LHR mRNA.luteinizing hormone receptor, microRNA, ovary, posttranscriptional regulation

Characterization of microRNAs differentially expressed during bovine follicle development

“…QPCR analyses confirmed that at least three of these miRNAs were also upregulated significantly in some or all of the large healthy follicle categories relative to large atretic follicles, thus identifying this set of miRNAs as very strong candidates with a role in the development of bovine dominant follicles. mir-144, mir-202, mir-451, and mir-652 were reported earlier to be expressed in the ovary (Hossain et al 2009, McBride et al 2012, Kitahara et al 2013, Schauer et al 2013, although their expression patterns within follicular tissues were not studied in detail. In this study, bta-miR-202 was restricted to the bovine gonad; moreover, its expression within the follicle was highest in granulosa cells while levels in ovarian stroma were negligible.…”

“…In addition to mir-202, biological roles for the clustered mir-144 and mir-451 have also been proposed, but only in relation to non-ovarian cells; those include regulatory roles in erythropoiesis (Pase et al 2009), differentiation of vascular smooth muscle cells (Turczyń ska et al 2013) and, potentially relevant during follicular differentiation, cholesterol trafficking in macrophages (Ramirez et al 2013). Interestingly, both miR-144 and miR-451, as well as miR-652, were upregulated in rat ovaries following an ovulatory hCG bolus, although no attempt was made to identify the specific cell type(s) involved (Kitahara et al 2013).…”

“…Several studies have already reported effects of specific miRNAs on different aspects of granulosa cell function , including steroidogenesis (Yao et al 2010, Xu et al 2011, Yin et al 2012, Dai et al 2013, proliferation (Yao et al 2010, Yan et al 2012, Dai et al 2013, survival (Carletti et al 2010, Yang et al 2012, terminal differentiation (Kitahara et al 2013), and cumulus expansion (Yao et al 2014). With a few exceptions (Carletti et al 2010, Kitahara et al 2013, most evidence on follicular roles of miRNAs has been obtained using cultured cells, particularly rodent cells, and in some cases actual changes in the expression of these miRNAs during follicle development have not been demonstrated.…”

“…Several studies have already reported effects of specific miRNAs on different aspects of granulosa cell function , including steroidogenesis (Yao et al 2010, Xu et al 2011, Yin et al 2012, Dai et al 2013, proliferation (Yao et al 2010, Yan et al 2012, Dai et al 2013, survival (Carletti et al 2010, Yang et al 2012, terminal differentiation (Kitahara et al 2013), and cumulus expansion (Yao et al 2014). With a few exceptions (Carletti et al 2010, Kitahara et al 2013, most evidence on follicular roles of miRNAs has been obtained using cultured cells, particularly rodent cells, and in some cases actual changes in the expression of these miRNAs during follicle development have not been demonstrated. Indeed, although genome-wide miRNA analyses of whole ovarian tissues (Landgraf et al 2007, Ro et al 2007, Mishima et al 2008, Hossain et al 2009, Ahn et al 2010, Juanchich et al 2013 or follicular, or luteal tissues (Fiedler et al 2008, McBride et al 2012, Miles et al 2012, da Silveira et al 2012, Donadeu & Schauer 2013, Schauer et al 2013, Sohel et al 2013 have been reported in several species, detailed spatiotemporal profiles encompassing several follicle developmental stages have not.…”

MicroRNA Regulation of Endocrine Functions in the Ovary

“…Another study performed using whole rat ovary homogenates identifi ed miRNA-136-3p as being overexpressed 6 h after hCG treatment and bioinformatic analyses indicated that 3′-UTR of the rat LHR has a miR-136-3p site within it. The biologic activity of miR-136 was then tested in primary rat granulosa cell culture and was shown to directly target the rat LHr (Kitahara et al 2013 ). It should be noted that bioinformatic (TargetScan) analysis of the human LHr 3′-UTR does not detect a miR-136-3p site within it.…”