Long non-coding RNAs in ovarian granulosa cells.

To identify differentially expressed lncRNAs in ovarian hyperstimulation syndrome (OHSS), which is characterized by enlarged ovaries and up-regulated vascular permeability, GCs were obtained from women with varying OHSS risks, for high-throughput sequencing. A total of 23,815 lncRNAs were detected and 482 of them showed differential expression: 205 lncRNAs were up-regulated and 277 lncRNAs were downregulated. Several ovarian biological processes were significantly involved in this phenomenon as demonstrated by KEGG pathway and GO analyses. Meanwhile, an lncRNA/miRNA interacting network was also established according to ceRNA regulatory mechanisms, a recent finding that adds to the complexities of miRNA-mediated gene modulation. ceRNAs are RNAs that mutually regulate other miRNAs via competitively binding the same miRNA recognition elements (MREs). In addition, expression screening identified eight novel lncRNAs (Supplementary Table  2 ) in GCs that were associated with risk factors for OHSS, suggesting that these lncRNAs might be potential participants in OHSS development [ 59 ]. The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes reproductive defects, such as anovulation and disorder of follicular steroidogenesis, in several mammals, including human, mouse, rat and pig [ 60 , 61 ]. In total, 1666 lncRNAs were characterized in TCDD-treated porcine GCs in vitro and 22 differentially expressed lncRNAs were identified. The potential functions of these 22 lncRNAs were predicted by analyzing their cis - and trans -regulated protein-coding genes. Two essential functional proteins of GCs, cytochrome P450 1A1 (CYP1A1) and aryl hydrocarbon receptor (AhR), were identified among the genes that were trans -regulated by the differentially expressed lncRNAs. The mRNA-lncRNA co-expression analysis showed that the TCDD-regulated lncRNAs might be involved in numerous cellular activities in GCs, such as cellular response to xenobiotics, proliferation, and dioxin metabolism. Therefore, these GC-specific lncRNAs may be involved in TCDD-induced reproductive defects [ 62 ]. Endometriosis and its surgical treatment have an adverse effect on the ovarian reserve and on oocyte development. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched abundant transcript 2 (NEAT2), is a well-studied lncRNA that is highly evolutionarily conserved [ 63 ] and is extensively investigated as an “onco-lncRNA” in various malignancies [ 64 , 65 ]. Its expression was downregulated in GCs (the isolated primary mouse GCs are mixed cumulus GCs and mural GCs) from patients with endometriosis. Moreover, MALAT1-knockdown repressed GCs proliferation via the induction of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, suggesting that the MALAT1 dysfunction might have an adverse effect on the development of oocytes in endometriosis [ 66 ]. Taken together, these data reflect the important role of GC-specific lncRNAs in a series of GC-related reproductive diseases, suggesting that GC-specific lncRNAs may be used as diagnostic markers or potential therapeutic targets for these diseases. However, the underlying mechanisms have not been systematically illuminated and these studies used in vitro assays. Therefore, to elucidate the specific function and mechanism of these lncRNAs in GCs obtained from patients suffering from ovarian diseases, advanced animal models and a large number of clinical samples will be required in subsequent studies.

To investigate the potential application of lncRNAs in clinical diagnosis and therapy of GC-related diseases, researchers currently focus on the function and underlying mechanisms of lncRNAs in GCs. High-throughput technologies, such as lncRNA sequencing, have been applied to screen lncRNAs in GCs in several pathological conditions. These differentially expressed lncRNAs may be useful for the diagnosis or prognosis of GC-associated diseases. Moreover, studies of individual lncRNAs in GCs highlight the potential use of lncRNAs as therapeutic targets for GC-related diseases. For example, encapsulated siRNA may be delivered to target specific lncRNAs for the treatment of GC-related diseases. Due to the complex spatial structure and unclear molecular regulatory mechanisms, studies of lncRNAs are still at a very early stage. Therefore, a deeper understanding of lncRNA functions in GCs will provide a promising foundation for a potential future use of lncRNAs in the diagnosis or treatment of GC-related diseases.

Granulosa cells are widely known for playing an essential role in both normal folliculogenesis and various ovarian disorders. LncRNAs exert their effects in GCs via multiple mechanisms. Until now, all regulatory mechanisms of lncRNA in GCs could be divided into five categories: 1) lncRNAs that regulate classical signaling pathways, such as lncRNA-LET (which induces the Wnt/β-catenin/Notch pathways), or lncRNA-SRA (which activates the NF-κB pathway) and lncRNA-MALAT1 and lncRNA-TUG1(which represses the ERK/MARK pathway) in GCs; 2) lncRNAs that activate transcription via activating their host genes or adjacent coding genes in GCs, including lncRNA-Amhr2, which activates the transcription of its neighboring gene Amhr2, or lncRNA-lncPrepþ96kb, which promotes its adjacent coding gene POP and HAS2, which was activated by its antisense lncRNA-HAS2-AS1; 3) ceRNA mechanism in GCs: PWRN2-miR-92b-3p-TMEM120B ceRNA network, LncRNA-LINC-01572:28/p27-SKP2-p27 ceRNA network and PVT1-miR-17-5p-PTEN ceRNA network in GCs; 4) P53-associated lncRNAs: lncRNA-BANCR and LncRNA-Meg3; 5) transcriptional factors-associated lncRNAs: lncRNA-HUPCOS interacts with RBPMS, lncRNA-CHBRP interacts with YY1 and SIX5 and lncRNA-HCP5 interacts with YB1(Table 2 ). The specific functional roles, upstream regulators, and downstream effectors of these dysregulated lncRNAs in GCs are still elusive. The dysregulated expression of lncRNAs in the circulation has been used as a biomarker in several studies that allowed distinguishing patients with ovarian disorders from healthy controls; however, the clinical usage of lncRNAs for ovarian disorder diagnosis remains to be established through large-cohort validation in different populations. In the future, advanced technologies, such as single-cell sequencing, CRISPR-Cas9, genetic animal models, and subsequent clinical trials must be employed to further elucidate the mechanisms and verify the clinical potential of lncRNAs in GCs as diagnostic or therapeutic targets for ovarian disorders. Table 2 Summary of regulatory mechanisms of lncRNAs in granulosa cells (GCs) Summary of regulatory mechanisms of lncRNAs in granulosa cells (GCs)

Granulosa cells (GCs) are somatic cells of the sex cord [ 1 ], which are associated with the development of oocytes in mammalian ovaries [ 2 , 3 ]. In addition, GCs are also implicated in various ovary-related diseases, including polycystic ovary syndrome (PCOS) [ 4 ], premature ovarian insufficiency (POI, also referred to as premature ovarian failure [POF]) [ 5 ], ovarian hyperstimulation syndrome (OHSS) [ 6 ], and GCs tumor (GCT) [ 7 ]. Previous studies have shown that genetic factors are involved in the development of these diseases [ 8 – 11 ]. Recent evidence has shown that several non-coding RNAs also affect these female reproductive system dysfunctions. Development of sequencing technology has led to in-depth genome and transcriptome analysis, which showed that over 85% of the human genome is transcribed [ 12 ]. However, the amount of protein products from RNA transcripts is very low compared to the overall number of transcripts, indicating that most RNA transcripts are non-coding. Such a large number of transcripts of non-coding RNAs (ncRNAs) suggests that ncRNAs play a more important and diverse role in biological processes than initially expected [ 13 , 14 ]. NcRNAs can be roughly divided into two groups: a group of short RNAs with a length less than 200 nucleotides long, such as microRNAs (miRNAs), small interfering RNA (siRNA), and piwi RNA (piRNA). The other category is long ncRNAs (lncRNAs) with a length longer than 200 nucleotides. Due to the lack of protein-coding capability, non-coding transcripts have been known as “junk DNA” or “transcriptional noise” for the past few decades [ 15 ]. However, many recent lncRNA-based studies have proved that lncRNAs have several functions and act via multiple regulatory mechanisms, including decoy, enhancer RNA, scaffold, guider, microRNA sponging and short peptides [ 16 , 17 ]. Long non-coding RNAs are classified based on their specific characteristics and their position relative to the host or adjacent protein-coding gene. Location-based classification of lncRNAs assigns them into categories such as antisense RNAs, long intergenic non-coding RNAs (lincRNAs), sense overlapping transcripts, sense intronic transcripts, and processed transcripts [ 18 ]. Characteristic-based classification, on the other hand, assigns them to categories such as lncRNA-activating (lncRNA-a) genes, pseudogenes, telomere-associated non-coding RNAs (TERRAs), transcribed ultraconserved regions (T-UCRs), enhancer RNAs (eRNAs), and circular RNAs [ 19 – 21 ]. Compared with the multitude of studies performed on miRNAs and protein-coding genes, we are still at a relatively early stage of investigating, naming, classifying, and identifying lncRNAs. Moreover, emerging studies have demonstrated that some lncRNAs affect the function of ovarian GCs and are thereby involved in both physiological conditions and pathological processes, such as human oocyte maturation, fertilization, embryo development, tumorigenesis, [ 22 ] and ovarian failure [ 23 , 24 ]. These studies have suggested that GC-specific lncRNAs could be considered as candidate diagnostic or prognostic markers, as well as treatment targets for various ovarian diseases [ 25 – 27 ]. In this review, we summarize the roles of lncRNAs in healthy and dysfunctional GCs and discuss the potential utilization of lncRNAs as diagnostic markers or treatment targets in clinical conditions.

Additional file 1. The fourteen overlapping lncRNAs that are transcribed from chromosome 2 and enhancer-like lncRNAs that were differently expressed in PCOS GCs. Additional file 2. The eight novel lncRNAs in GCs that were associated with risk factors for OHSS. Additional file 1. The fourteen overlapping lncRNAs that are transcribed from chromosome 2 and enhancer-like lncRNAs that were differently expressed in PCOS GCs. Additional file 2. The eight novel lncRNAs in GCs that were associated with risk factors for OHSS.