miRNA Profiling Reveals miRNA-130b-3p Mediates DENND1A Variant 2 Expression and Androgen Biosynthesis

Abstract

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of reproductive-age women involving overproduction of ovarian androgens and, in some cases, from the adrenal cortex. Family studies have established that PCOS is a complex heritable disorder with genetic and epigenetic components. Several small, noncoding RNAs (miRNAs) have been shown to be differentially expressed in ovarian cells and follicular fluid and in the circulation of women with PCOS. However, there are no reports of global miRNA expression and target gene analyses in ovarian theca cells isolated from normal cycling women and women with PCOS, which are key to the elucidation of the basis for the hyperandrogenemia characteristic of PCOS. With the use of small RNA deep sequencing (miR-seq), we identified 18 differentially expressed miRNAs in PCOS theca cells; of these, miR-130b-3p was predicted to target one of the PCOS genome-wide association study candidates, differentially expressed in neoplastic vs normal cells domain containing 1A (DENND1A). We previously reported that DENND1A variant 2 (DENND1A.V2), a truncated isoform of DENND1A, is upregulated in PCOS theca cells and mediates augmented androgen biosynthesis in PCOS theca cells. The comparison of miR-130b-3p in normal and PCOS theca cells demonstrated decreased miR-130b-3p expression in PCOS theca cells, which was correlated with increased DENND1A.V2, cytochrome P450 17α-hydroxylase (CYP17A1) mRNA and androgen biosynthesis. miR-130b-3p mimic studies established that increased miR130b-3p is correlated with decreased DENND1A.V2 and CYP17A1 expression. Thus, in addition to genetic factors, post-transcriptional regulatory mechanisms via miR-130b-3p underly androgen excess in PCOS. Ingenuity® Pathway Analysis Core Pathway and Network Analyses suggest a network by which miR-130b-3p, DENND1A, the luteinizing hormone/choriogonadotropin receptor, Ras-related protein 5B, and signaling pathways that they potentially target may mediate hyperandrogenism in PCOS.

Colocalization of Polycystic Ovary Syndrome Candidate Gene Products in Theca Cells Suggests Novel Signaling Pathways

“…We found that DENND1A.V2 expression is elevated in theca cells of women with PCOS [16–18]. Moreover, forced expression of DENND1A.V2 in normal human theca cells in culture resulted in increased expression of CYP17A1 and production of androgens, whereas knockdown of DENND1A.V2 mRNA in PCOS theca cells reduced CYP17A1 expression and androgen secretion, supporting the notion that DENND1A.V2 contributes to hyperandrogenism in PCOS [16].…”

“…The studies of genetic and molecular factors involved in the pathophysiology of the PCOS accelerated after milestone GWAS identified PCOS candidate loci, including those near DENND1A , INSR , YAP1 , C9orf3 , RAB5B , HMGA2 , TOX3 , SUMO1P1/ZNF217 , THADA , and LHCGR [6–10]. In our previous studies, we established that a truncated splice variant of PCOS GWAS candidate gene, DENND1A , termed DENND1A.V2, showed increased expression in PCOS theca cells [16–18]. DENND1A has been described as a clathrin-binding protein that most likely is related to gonadotropin receptor signaling as part of the clustering and endocytosis of plasma membrane receptors via the involvement of RAB5 proteins [50, 51].…”

“…LH stimulation is known to be required for the excess ovarian androgen levels in PCOS, whereas RAB5 proteins, particularly RAB5A, are known to play a role in endocytosis and gonadotropin receptor signaling [3, 19]. We have previously proposed that these PCOS GWAS loci/genes, along with DENND1A , form a network that contributes to the hyperandrogenemia of PCOS, with DENND1A.V2 being the link between LHCGR accumulated in coated pits and the trafficking of endocytic vesicles containing LHCGR and its downstream signaling molecules, including RAB5B [18, 20].…”

Genetic Susceptibility to Joint Occurrence of Polycystic Ovary Syndrome and Hashimoto’s Thyroiditis: How Far Is Our Understanding?

“…Recently, a functional network, including miR-130b-3p, DENND1A.V.2, LHCGR, RSB5B, and the signaling pathways they target, was proposed to potentially mediate PCOS-related hyperandrogenemia. The functional significance of this network is strongly supported by the discovery of the co-localization of LHCGR, DENND1A.V.2, and RAB5B proteins in theca cells ( 82 ), as well as the fact that decreased expression of miR-130b-3p, which is predicted to target DENND1A, correlates with increased V2 mRNA levels and androgen synthesis in PCOS theca cells ( 83 ).…”

Non-coding RNAs in polycystic ovary syndrome: a systematic review and meta-analysis

“…Eight studies have revealed that expression of miRNAs was altered in other samples of PCOS population, including blastocysts [ 69 ], adipose tissue [ 26 , 70 ], ovarian theca cells [ 73 ], ovarian theca interna tissues [ 17 ], ovary cortex [ 62 , 71 ] and ovarian tissue [ 72 ]. McCallie et al [ 69 ] reported that blastocysts isolated from patients with PCOS exhibited significantly decreased expression of six miRNAs (miR-let-7a, miR-19a, miR-19b, miR-24, miR-92, and miR-93) in comparison with donor fertile control blastocysts.…”