NCBI Summary:
microRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA. The RefSeq represents the predicted microRNA stem-loop. [provided by RefSeq, Sep 2009]
Involvement of miRNAs in ovarian follicular and luteal development. Donadeu FX et al. Although much progress has been made in the genetic dissection of biological networks involved in follicular/luteal development in the mammalian ovary, the gene regulation mechanisms involved are still poorly understood. Over the last 10 years, miRNAs have emerged as master regulators of tissue growth and differentiation in animals. However, compared to other body tissues, little is still known on the functional involvement of miRNAs in the ovary. Several studies have identified miRNA populations specifically associated with the development of follicles and corpora lutea, particularly in relation to the follicular-luteal transition, and the functional involvement of some of these miRNAs has been characterised in vitro and/or in vivo. Specifically, three different miRNAs, miR-224, miR-378 and miR-383, have shown to be involved in regulating aromatase expression during follicle development. In addition, miR-21 has been identified as promoting follicular cell survival during ovulation, and pro-angiogenic miR-17-5p and let-7b were shown to be necessary for normal development of the corpus luteum. Experimental evidence for the involvement of several other miRNAs in different aspects of follicle/luteal development has also been obtained. In addition, many of these studies exemplify the challenges associated with identifying physiologically relevant targets of ovarian miRNAs. Continuous advances in this field will be considerably facilitated by progress in understanding miRNA physiology in other body systems and will eventually lead to a much better understanding of the control of follicular/luteal development. In turn, through the potential offered by miRNA diagnostics and miRNA therapeutics, this new knowledge should bring considerable benefits to Reproductive medicine. review
Cellular localization
Nuclear
Comment
Differential Expression of miR-93 and miR-21 in Granulosa Cells and Follicular Fluid of Polycystic Ovary Syndrome Associating with Different Phenotypes. Naji M et al. (2017) The heterogeneous and multifactorial essence of polycystic ovary syndrome (PCOS) renders a remarkable significance to microRNAs (miRNAs). Normo-androgenic (NA) and hyperandrogenic (HA) PCOS patients were compared with matched healthy women. Expression of miRNAs and TGFβ signaling genes was studied by qRT-PCR and western blotting. Effect of androgen on expression of miR-93 and miR-21 and involvement of androgen receptor were appraised. In granulosa cells (GCs), miR-93 and miR-21 showed significantly increased levels in HA patients compared to NA patients. On the contrary, follicular fluid (FF) levels of both miRNAs were significantly decreased in HA group compared to control women. No significant change in the expression of miRNAs in serum samples was detected. Furthermore, mRNA levels of SMAD7 and TGFBR2 were significantly downregulated in GCs of HA group compared to NA and control subjects. TGFBR2 protein level was significantly decreased in HA patients compared to controls. Free testosterone and free androgen index were positively correlated with expression of miR-93 and miR-21 in GCs of PCOS group. Our findings show distinct molecular signature of different subtypes of PCOS. Intermediary position of miRNAs as androgen responsive factors may play critical role in the pathogenesis of PCOS in hyperandrogenic condition.//////////////////
Ciculating miRNA-21 as a Biomarker Predicts Polycystic Ovary Syndrome (PCOS) in Patients. Jiang L et al. (2015) Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, hyperinsulinemia, and infertility. In PCOS, abnormal regulation of relevant genes is required for follicular development. By binding to the 3' untranslated region (3'URT), microRNAs (miRNAs) are widely involved in posttranscriptional gene regulation. However, few studies have been conducted on circulating miRNA expression in PCOS. This study aims to describe altered expression of circulating miR-21 in PCOS. The expression of serum miRNAs of PCOS patients were explored using the TaqMan Low Density Array followed by individual quantitative reverse transcription polymerase chain reaction assays. The protein level of LATS1 was determined using Western blot. To validate whether miR-21 targeted LATS1, the luciferase assay was applied. In comparison with normal subjects, the circulating level of miRNA-21 was significantly enhanced in PCOS patients. In PCOS patients, the expression levels of MST1/2, LATS1/2, TAZ were much lower than the control subjects. Luciferase reporter assay revealed that LATS1 was a downstream target of miR-21. In comparison with normal subjects, serum miR-21 is obviously increased in PCOS patients. Through targeting LATS1, miR-21 could prompt PCOS progression and could act as a novel non-invasive biomarker for diagnosis of PCOS.//////////////////
miRNA-21-3p targeting of FGF2 suppresses autophagy of bovine ovarian granulosa cells through AKT/mTOR pathway. Ma L et al. (2020) It is widely thought that the main reason for ovarian follicular atresia is apoptosis of granulosa cells, however, accumulating evidence suggests that autophagy plays a role in the fate of granulosa cells. Although epigenetic regulation including miR-21-3p associated with autophagy process has been reported in many cancer types, nevertheless, the mechanism of miR-21-3p in bovine ovary is poorly understood. In the present study, bovine ovarian granulosa cells (BGCs) were used as a model to elucidate the autophagy and role of miR-21-3p in a cattle ovary. The results from gene expression and tagged autophagosomes showed the autophagy in BGCs and miR-21-3p was identified as an important miRNA regulating autophagy of BGCs. The current results indicated that FGF2 was a validated target of miR-21-3p in autophagy regulation of BGCs according to the results from FGF2 luciferase reporter assays and FGF2 overexpression (oe-FGF2) or small interference (si-FGF2). Transfection of miR-21-3p mimic and si-FGF2 plasmids resulted in decreasing phosphorylated AKT and mTOR, while transfection of miR-21-3p inhibitor and oe-FGF2 increased the phosphorylated level of AKT and mTOR in BGCs. These data indicate that regulation of miR-21-3p on BGCs autophagy through AKT/mTOR pathway. In summary, this study suggests that miR-21-3p targets FGF2 to inhibit BGCs autophagy by repressing AKT/mTOR signaling.//////////////////
MicroRNA-21 as a regulator of human cumulus cell viability and its potential influence on the developmental potential of the oocyte. Bartolucci AF et al. (2020) MicroRNA-21 ( MIR-21 ) is expressed in bovine, murine and human cumulus cells with its expression in murine and bovine cumulus cells correlated with oocyte developmental potential. The aim of this study was to assess the relationship between cumulus cell MIR-21 and human oocyte developmental potential. These studies revealed that both the immature and mature forms of MIR-21 ( MIR-21-5p ) were elevated in cumulus cells of oocytes that developed into blastocysts compared to cumulus cells of oocytes that arrested prior to blastocyst formation. This increase in MIR-21 was observed regardless of whether the oocytes developed into euploid or aneuploid blastocysts. Moreover, MIR-21-5p levels in cumulus cells surrounding oocytes that either failed to mature or matured to metaphase II but failed to fertilize, were ≈ 50% less than the MIR-21-5p levels associated with oocytes that arrested prior to blastocyst formation. Why cumulus cells associated with oocytes of reduced developmental potential expressed less MIR-21-5p is unknown. It is unlikely due to reduced expression of either 1) the receptors of Growth Differentiation Factor 9 (GDF9) or 2) Drosha Ribonuclease III (DROSHA) and Dicer Ribonuclease III (DICER) which sequentially promote the conversion of immature forms ofMIR-21to matureMIR-21. Furthermore, cultured cumulus cells treated with aMIR-21-5pinhibitor had an increase in apoptosis and a corresponding increase in the expression ofPTEN, a gene known to inhibit the AKT-dependent survival pathway in cumulus cells. These studies provide evidence for a role ofMIR-21in human cumulus cells that influences the developmental potential of human oocytes.////////////////// miR-21-3p inhibits autophagy of bovine granulosa cells by targeting VEGFA via PI3K/AKT signaling. Ma L et al. (2019) It is well documented that granulosa cell apoptosis is the main reason for follicular atresia and death, however, increasing evidence suggests that autophagy plays an important role in the fate of granulosa cells. MiR-21-3p regulated many fundamental biological processes and was pivotal in autophagy of tumor cell, nevertheless, the autophagy in cattle ovary and how miR-21-3p regulates the follicular cells is unknown. In this study, we aimed to elucidate the autophagy and the role of miR-21-3p in cattle ovary using bovine primary ovarian granulosa cells (BGCs). The results showed the autophagy for the first time in BGCs in large follicle according to autophagic gene transcript of LC3, BECN-1, ATG3, protein expression of LC3, P62 and LC3 puncta, a standard marker for autophagosomes. MiR-21-3p was identified as a novel miRNA that repressed BGCs autophagy according to the results from plasmids transfection of miR-21-3p mimics and inhibitor. Meanwhile, VEGFA was confirmed to be a validated target of miR-21-3p in BGCs using luciferase reporter assays and the results of VEGFA expression decreased with transfection of miR-21-3p mimics, while increased with transfection of miR-21-3p inhibitor. In addition, small interference mediated knockdown of VEGFA significantly inhibits BGCs autophagy signaling, however, overexpression of VEGFA in BGCs promoted autophagy in the presence of miR-21-3p. Finally, the results of AKT and it's phosphorylation suggested that miR-21-3p suppressed VEGFA expression through downregulating AKT phosphorylation signaling. In summary, this study demonstrates that miR-21-3p inhibits BGCs autophagy by targeting VEGFA and attenuating PI3K/AKT signaling.//////////////////
Characterization of the miRNA regulators of the human ovulatory cascade. Yerushalmi GM et al. (2018) Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified differentially expressed mRNAs between human cumulus granulosa cells (CGCs) from immature early antral follicles (germinal vesicle - GV) and mature preovulatory follicles (metaphase II - M2). In this study, we performed an integrated analysis of the transcriptome and miRNome in CGCs obtained from the GV cumulus-oocyte complex (COC) obtained from IVM and M2 COC obtained from IVF. A total of 43 differentially expressed miRNAs were identified. Using Ingenuity IPA analysis, we identified 7288 potential miRNA-regulated target genes. Two hundred thirty-four of these target genes were also found in our previously generated ovulatory gene library while exhibiting anti-correlated expression to the identified miRNAs. IPA pathway analysis suggested that miR-21 and FOXM1 cooperatively inhibit CDC25A, TOP2A and PRC1. We identified a mechanism for the temporary inhibition of VEGF during ovulation by TGFB1, miR-16-5p and miR-34a-5p. The linkage bioinformatics analysis between the libraries of the coding genes from our preliminary study with the newly generated library of regulatory miRNAs provides us a comprehensive, integrated overview of the miRNA-mRNA co-regulatory networks that may play a key role in controlling post-transcriptomic regulation of the ovulatory process.////////////////// MicroRNA 21 Blocks Apoptosis in Mouse Periovulatory Granulosa Cells. Carletti MZ et al. MicroRNA (miRNA) play important roles in many developmental processes including cell differentiation and apoptosis. Transition of proliferative ovarian granulosa cells to terminally differentiated luteal cells in response to the ovulatory surge of luteinizing hormone (LH) involves rapid and pronounced changes in cellular morphology and function. MicroRNA 21 (miR-21, official symbol Mir21) is one of three highly LH-induced miRNA in murine granulosa cells, and here we examine the function and temporal expression of Mir21 within granulosa cells as they transition to luteal cells. Granulosa cells were transfected with blocking (2'-O-methyl) and locked nucleic acid (LNA-21) oligonucleotides and mature Mir21 expression decreased to 1/9th and 1/27th of its basal expression, respectively. LNA-21 depletion of Mir21 activity in cultured granulosa cells induced apoptosis. In vivo, follicular granulosa cells exhibit a decrease in cleaved caspase 3, a hallmark of apoptosis, 6 h after the LH/hCG surge, coincident with the highest expression of mature Mir21. To examine whether Mir21 is involved in regulation of apoptosis in vivo, mice were treated with a phosphothioate modified LNA-21 oligonucleotide and granulosa cell apoptosis examined. Apoptosis increased in LNA-21 treated ovaries and ovulation rate decreased in LNA-21 treated ovaries, compared to their contralateral controls. We have examined a number of Mir21 apoptotic target transcripts identified in other systems; currently none of these appear to play a role in the induction of ovarian granulosa cell apoptosis. This study is the first to implicate the anti-apoptotic Mir21 (an oncogenic miRNA) as playing a clear physiologic role in normal tissue function.
Expression regulated by
LH
Comment
CiRS-126 inhibits proliferation of ovarian granulosa cells through targeting the miR-21-PDCD4-ROS axis in a polycystic ovarian syndrome model. Lu J et al. (2020) Polycystic ovarian syndrome (PCOS) is considered to be one of the most prevalent endocrine disorders affecting women of reproductive age. CiRS-126, an innovative circular microRNA, has previously been proven to be a promising miR-21 sponge. However, a proper understanding of the impact of ciRS-126 on PCOS is needed. Circular RNA (CiRS) profiles were initially evaluated in ovarian cortex samples obtained from 18 women with PCOS as well from 9 women without PCOS. Insulin-induced ovarian granulosa cells isolated from mice were utilized for the functional study. CiRS microarray analysis and quantitative real-time PCR indicated that ciRs-126 expression was downregulated while miR-21 expression was upregulated in PCOS samples and insulin-induced granulosa cells as compared with non-PCOS samples and non-insulin-induced granulosa cells. Furthermore, ectopic overexpression of ciRS-126 was associated with a reduction in proliferation and increased apoptosis in insulin-treated granulosa cells. Meanwhile, bioinformatic prediction and the results of the dual-luciferase reporter assay indicated the presence of consecutive binding in the ciRS-126-miR-21-programmed cell death protein 4 (PDCD4) axis. Moreover, overexpression of miR-21 blocked ciRS-126 repression of proliferation and triggered the death of insulin-induced granulosa cells. Excessive PDCD4 expression counteracted the influence of miR-21 on cell death and proliferation. The data indicated that PDCD4 played a regulatory role in ROS generation, which is reportedly involved in apoptosis. Therefore, ciRS-126 reduction in PCOS granulosa cells targeted the miR-21-PDCD4 axis to reduce proliferation and promote apoptosis. CiRS-126 shows potential as a promising predictor of clinical outcome as well as a therapeutic target in PCOS.//////////////////
STAT3 signaling stimulates miR-21 expression in bovine cumulus cells during in vitro oocyte maturation. Tscherner A et al. (2018) MicroRNAs are potent regulators of gene expression that have been widely implicated in reproduction and embryo development. Recent studies have demonstrated that miR-21, a microRNA extensively studied in the context of disease, is important in multiple facets of reproductive biology including folliculogenesis, ovulation, oocyte maturation and early mammalian development. Surprisingly, little is known about the mechanisms that regulate miR-21 and no studies have characterized these regulatory pathways in cumulus-oocyte complexes (COCs). We therefore investigated miR-21 in an in vitro model of bovine oocyte maturation. Levels of the primary transcript of miR-21 (pri-miR-21) and mature miR-21 increased markedly in COCs over the maturation period. Cloning of the bovine pri-miR-21 gene and promoter by 5'3'RACE (rapid amplification of cDNA ends) revealed a highly conserved region immediately upstream of the transcription start site and two alternatively-spliced variants of pri-miR-21. The promoter region contained several putative transcription factor binding sites, including two for signal transducer and activator of transcription 3 (STAT3). Mutation of these sites significantly decreased both the intrinsic activity of pri-miR-21 promoter-luciferase constructs and the response to leukemia inhibitory factor (LIF) (a STAT3 activator) in cultured MCF7 cells. In COCs, treatment with a STAT3 pathway inhibitor markedly decreased pri-miR-21 expression and prevented cumulus expansion. Pri-miR-21 expression was also inhibited by the protein synthesis inhibitor cycloheximide, suggesting that a protein ligand or signaling cofactor synthesized during maturation is necessary for transcription. Together these studies represent the first investigation of signaling pathways that directly influence miR-21 expression in bovine oocytes and cumulus cells.//////////////////
Ovarian localization
Oocyte, Granulosa
Comment
PPARγ is regulated by miR-27b-3p negatively and plays an important role in porcine oocyte maturation. Song C et al. (2016) To elucidate the key miRNAs and the signalling pathways that are involved in porcine oocyte maturation, we performed a deep sequencing analysis of the miRNAs of pig germinal vesicle (GV) oocytes and metaphase II (MII) oocytes. Seven differentially expressed (DE) miRNAs were identified and the expression levels of miR-21 and miR-27b-3p were further confirmed by QPCR analysis. The target genes of 7 DE miRNAs were predicted and subjected to pathway analysis. Interestingly, fatty acid metabolism and fatty acid biosynthesis were the top two significantly enriched molecular functions during oocyte maturation. Heat map, which was built with 7 DE miRNAs and the enriched the molecular functions, revealed that miR-21, miR-27b-3p, miR-10a-5p and miR-10b-5p were involved in fatty acid metabolism. In particular, the regulatory role of miR-27b-3p on peroxisome proliferator-activated receptor-γ (PPARγ) was confirmed by their inversed expression patterns in GV and MII oocytes and luciferase report assays. In addition, we observed that PPARγ agonist (rosiglitazone) treatment significantly enhanced porcine oocyte maturation rate and early embryo developmental competent. Taken together, our results demonstrated that miR-27b and its target, PPARγ, play the vital roles in pig oocyte maturation through regulating the fatty acid metabolism. These data increased our understanding of the regulatory gene networks in porcine oocyte maturation and development.//////////////////
Mir-21 Is Enriched in the RNA-Induced Silencing Complex and Targets col4a1 in Human Granulosa Cell Lines. Mase Y et al. MicroRNAs (miRNAs) are noncoding small RNAs that play important roles in a variety of physiological and pathological events. In this study, we performed large-scale profiling of EIF2C2-bound miRNAs in 3 human granulosa-derived cell lines (ie, KGN, HSOGT, and GC1a) by high-throughput sequencing and found that miR-21 accounted for more than 80% of EIF2C2-bound miRNAs, suggesting that it was enriched in the RNA-induced silencing complex (RISC) and played a functional role in human granulosa cell (GC) lines. We also found high expression levels of miR-21 in primary human GCs. Assuming that miR-21 target mRNAs are enriched in RISC, we performed cDNA cloning of EIF2C2-bound mRNAs in KGN cells. We identified COL4A1 mRNA as a miR-21 target in the GC lines. These data suggest that miR-21 is involved in the regulation of the synthesis of COL4A1, a component of the basement membrane surrounding the GC layer and granulosa-embedded extracellular structure.
Follicle stages
Preovulatory, Corpus luteum
Comment
Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary. McBride D et al. Little is known about the involvement of microRNAs (miRNAs) in the follicular-luteal transition. The aim of this study was to identify genome-wide changes in miRNAs associated with follicular differentiation in sheep. miRNA libraries were produced from samples collected at defined stages of the ovine oestrous cycle and representing healthy growing follicles (diameter, 4.0-5.5 mm) pre-ovulatory follicles (6.0-7.0 mm), early corpora lutea (day 3 post-oestrus) and late corpora lutea (day 9). A total of 189 miRNAs reported in sheep or other species and an additional 23 novel miRNAs were identified by sequencing these libraries. miR-21, miR-125b, let-7a and let-7b were the most abundant miRNAs overall, accounting for 40% of all miRNAs sequenced. Examination of changes in cloning frequencies across development identified nine different miRNAs which expression decreased in association with the follicular-luteal transition and eight miRNAs which expression increased during this transition. Expression profiles were confirmed by Northern analyses, and experimentally validated targets were identified using miRTarBase. A majority of the 29 targets identified represented genes known to be actively involved in regulating follicular differentiation in vivo. Finally, luteinisation of follicular cells in vitro resulted in changes in miRNA levels that were consistent with those identified in vivo, and these changes were temporally associated with changes in levels of putative miRNA targets in granulosa cells. In conclusion, this is the first study to characterise genome-wide miRNA profiles during different stages of follicle and luteal development. Our data identifies a subset of miRNAs which are potentially important regulators of the follicular-luteal transition.