microRNA miR-513a-3p acts as a co-regulator of luteinizing hormone/chorionic gonadotropin receptor gene expression in human granulosa cells
Introduction
The two closely related glycoprotein hormones, luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are crucial for male and female reproductive processes. The action of LH and hCG is mediated by the luteinizing hormone/chorionic gonadotropin receptor (LHCGR), which belongs to the superfamily of G protein coupled receptors and is expressed on the cellular surface of Leydig cells within the testes and theca cells, differentiated granulosa cells (GCs) and luteal cells of the ovary (Hiort, 2000, Ascoli et al., 2002). In the follicular phase of the female menstrual cycle, LH acts on theca cells and stimulates the production of androgen precursors. In theca cells the LHCGR is consistently expressed whereas in GCs, LHCGR expression is increased by FSH and estradiol in small- and medium-sized follicles (Channing et al., 1980). Hence, in the second half of the follicular phase LH acts on GCs to activate the production of estrogens and to stimulate the proliferation of GCs (Zeleznik, 2001, Lindeberg et al., 2007, Young and McNeilly, 2010). During its midcycle surge, LH induces follicular changes important for maturation, ovulation and luteinization (Conti et al., 2012). Besides the well-known desensitisation and internalisation processes, the regulation of the dynamically alternating LHCGR expression has been studied extensively and different levels of regulation have been identified. The impact of transcription factors and epigenetic modifications, as well as mechanisms based on regulatory proteins interacting with the LHCGR mRNA or protein have been demonstrated (Kash and Menon, 1998, Geng et al., 1999, Zhang and Dufau, 2000, Zhang and Dufau, 2002, Zhang et al., 2005, Dufau et al., 2010, Menon and Menon, 2012, Kogure et al., 2013). Post-transcriptional regulation of the spatial and temporal LHCGR gene expression presents an important element for the dynamic expression changes necessary for normal physiological function during the female cycle. Post-transcriptional gene regulation includes processes such as RNA editing, mRNA transport/localization, events associated with the initiation and regulation of translation/degeneration and alternative splicing of mRNA.
The human LHCGR consists of 12 exons and is therefore prone to extensive alternative splicing (Rousseau-Merck et al., 1990, Kossack et al., 2008). Differently spliced variants of the LHCGR are found in human reproductive tissues and cells including testis, ovary and GCs (Minegishi et al., 1997, Minegishi et al., 2007, Madhra et al., 2004, Kossack et al., 2008, Fowler et al., 2009). The LHCGR splice variant lacking exon 9 was shown to interact with full-length gonadotropin receptors, thereby playing a putative role in regulating gonadotropin receptor expression and function in the human ovary (Minegishi et al., 1997). However, the specific physiological and clinical roles of the multitude of splice variants are unknown so far and the majority of them are trapped within the cell. Furthermore, a process of post-transcriptional regulation of the LHCGR expression was detected in ovarian GCs, linking the increased degradation of the LHCGR transcript after stimulation with a pharmacological dose of hCG to mevalonate kinase (MVK). MVK is an enzyme in the cholesterol biosynthetic pathway that also acts as an RNA-binding protein (Kash and Menon, 1998, Menon et al., 2011). In rat GCs an up-regulation of Lhr mRNA based on an increase in Lhr mRNA stability occurred in the presence of estradiol and FSH mediated by decreasing Mvk levels (Ikeda et al., 2008). Furthermore, microRNAs (miRNAs) have been shown to play a role in the regulation of the Lhr mRNA expression in rat primary granulosa cells (Kitahara et al., 2013). Apart from the known post-transcriptional regulatory processes, additional mechanisms of LHCGR regulation at this level might additionally also exist in the human. The impact of additional post-transcriptional mechanisms such as regulation by miRNAs may well add a novel layer of complexity to the regulatory network of LHCGR gene expression and thereby increase the flexibility and precision of functional regulation.
The large number of miRNAs expressed in reproductive tissues indicates that this form of post-transcriptional gene regulation plays a role in various aspects of reproductive physiology (Carletti and Christenson, 2009, Papaioannou and Nef, 2010, Donadeu et al., 2012, Hossain et al., 2012b, Nothnick, 2012). miRNAs comprise a class of single-stranded, endogenous, non-coding small RNAs that post-transcriptionally regulate gene expression by partial, complementary base-pairing to specific target mRNAs (Lagos-Quintana et al., 2001, Bartel, 2004, Kim, 2005). In order to understand the general impact of miRNA-mediated gene regulation in female reproduction the role of Dicer, a cytosolic, multi-domain protein required for miRNA processing, has been investigated using knockout mouse models (Carletti and Christenson, 2009, Luense et al., 2009). Studies revealed that the loss of Dicer within different reproductive tissues was associated with developmental arrest and female infertility (Bernstein et al., 2003, Murchison et al., 2007, Nagaraja et al., 2008, Gonzalez and Behringer, 2009, Mattiske et al., 2009). Many studies have used GCs as a powerful in vitro model for gain- and loss-of-function approaches to analyse the effects of specific miRNAs (Fiedler et al., 2008, Carletti et al., 2010, Yao et al., 2010, Xu et al., 2011, Yin et al., 2012). Thereby, miR-503 was shown to have a gonadotropin-induced function regarding the regulation of follicle granulosa cell proliferation and differentiation, and especially luteinization (Lei et al., 2010). Using human ovarian cancer cells (SKOV3) Cui et al. analysed miRNA expression and regulation by LH, demonstrating that LH regulates miRNA expression in LHR + SKOV3 cells (Cui et al., 2011). Although miRNAs have been implicated in hormonal regulation and their expression has been shown to vary in reproductive tissues in response to pituitary and gonadal hormones (Fiedler et al., 2008, Yao et al., 2010, Hossain et al., 2012b), the effects of miRNAs on hormone receptor expression in human ovarian GCs have not yet been analysed in detail.
As the understanding of the role of miRNAs in post-transcriptional receptor gene regulation can provide novel insights into gonadotropin-related functions, we studied miRNAs as a putative mechanism regarding the regulation of the dynamic spatio-temporal expression of the LHCGR. By computational prediction tools applying stringent criteria we identified miR-513a-3p, which targets the 3′UTR of the LHCGR. In vitro experiments as well as ex vivo studies with primary granulosa cells revealed the regulatory impact of miR-513a-3p on LHCGR expression, highlighting a so far unknown mechanism of regulation at the post-transcriptional level.
Section snippets
Computational prediction of miRNAs targeting the LHCGR
The LHCGR gene was screened for putative miRNA target sequences using the programs TargetScanHuman and miRanda (John et al., 2004, Lewis et al., 2005; http://www.targetscan.org; http://www.microrna.org).
Real-time PCR
Relative gene expression of LHCGR and miR-513a-3p was investigated employing quantitative PCR analysis. RNA was isolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription of 100 ng of total RNA was performed using iScript (Bio-Rad Laboratories, Muenchen, Germany) according
Results
The LHCGR displays several characteristics that suggest a stringent regulation by miRNAs such as a 3′UTR of medium length (Geng et al., 1999, Cheng et al., 2009) as well as high content of CG-dinucleotides in the promoter region (Zhang et al., 2005, Sinha et al., 2008).
Discussion
Follicle development requires the coordinated interaction of the oocyte and the surrounding layers of somatic granulosa and theca cells, which is tightly regulated by a multitude of genes including the LHCGR. In this study, we present miRNAs as an additional element in the regulatory circuits of LHCGR expression. Thereby, we provide novel insights into the spatio-temporal coordinated expression of the LHCGR gene and extend knowledge with respect to the function of the LHCGR for fertility and
Authors’ roles
All authors approved the final version to be published. B.T. contributed to study conception, acquisition, analysis and interpretation of data and prepared the manuscript. N.K. contributed to study conception and manuscript preparation. V.N. contributed to clinical characterisation of patients, acquisition of material and revised the manuscript. A.N.S. contributed to clinical conception of the study, recruitment of patients and revised the manuscript. J.G. contributed to study conception,
Funding
This work was supported by the German research foundation (GR 1547/13-1 and SCHL 394/9-1) and an IMF grant (NO 1 1 12 12).
Acknowledgment
We thank R. Sandhowe, L. Lahrmann and N. Terwort for invaluable technical assistance.
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