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]
General function
RNA binding
Comment
Cellular localization
Cytoplasmic
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Ovarian function
Ovulation, Steroid metabolism, Early embryo development
Comment
Doxorubicin induces cytotoxicity and miR-132 expression in granulosa cells. Al-Kawlani B et al. (2020) Doxorubicin (DOX) is one of the most commonly used drugs for the treatment of childhood cancers, including leukemia and lymphomas. Despite the high survival rate, female leukemia survivors are at higher risk of ovarian failure and infertility later in life. Treatment with chemotherapeutic drugs like DOX is associated with damage in ovarian follicles, but the affectation grade of granulosa cells remains unclear. To assess and avoid the possible side-effects of DOX, early biomarkers of ovarian injury and chemotherapy-induced ovarian toxicity should be identified. MicroRNAs (miRNAs) have emerged in recent years as a promising new class of biomarkers for drug-induced tissue toxicity. In this study, the effects of DOX on cell viability, steroidogenesis, and miRNA expression were studied in primary granulosa cells (GCs) and in two cellular models (COV434 and KGN cells). We report that compared to other chemotherapeutic drugs, DOX treatment is more detrimental to granulosa cells as observed by decrease of cell viability. Treatment with DOX changes the expression of the aromatase gene (CYP19A1) and the secretion of 17β-estradiol (E2) in a cell-specific manner. miR-132-3p is dose-dependently increased by DOX in all cellular models. In absence of DOX, miR-132-3p overexpression in COV434 cells has no effect on E2 secretion or CYP19A1 expression. Altogether, these findings contribute to understanding the hormonal disbalance caused by DOX in human ovarian cells and suggest miR-132 as a putative sensor to predict DOX-induced ovarian toxicity.//////////////////
Regulation of adrenal and ovarian steroidogenesis by miR-132. Hu Z et al. (2017) miR-132 is hormonally regulated in steroidogenic cells of the adrenal gland, ovary and testis. Here, we examined the potential role of miR-132 in the control of steroidogenesis. Transfection of Y1 adrenal cells with miR-132 increased mRNAs of 3β-HSD and 20α-HSD enzymes, which catalyze the sequential conversion of pregnenolone to progesterone to biologically inactive 20α-OHP. Overexpression of miR-132 reduced MeCP2 and StAR protein expression, basal progestin (progesterone and 20α-OHP) production, but enhanced their production in response to cAMP stimulation. Use of 3H] pregnenolone and free-diffusible 22(R)-hydroxycholesterol further confirmed that miR-132 promotes the production of 20α-OHP by upregulating the 3β-HSD and 20α-HSD. Evidence is also presented that StAR is a direct target of miR-132. Transient transfection of Y1 cells with miR-132 demonstrated that miR-132 induction of 3β-HSD and 20α-HSD was accompanied by significant suppression of one of its target gene products, MeCP2. In contrast, co-expression of miR-132 plus MeCP2 protein partially blocked the ability of miR-132 to upregulate the expression and function of 3β-HSD and 20α-HSD. Moreover, suppression of MeCP2 protein with SiRNA resulted in increased the expression of 3β-HSD and 20α-HSD, further demonstrating that miR-132 induces the expression of these two enzymes via inhibition of MeCP2. Likewise, overexpression of miR-132 increased 20α-OHP production with and without HDL loading, while knockdown of miR-132 resulted in a significant decrease of 20α-OHP production by the granulosa cells. In conclusion, our data suggest that miR-132 attenuates steroidogenesis by repressing StAR expression and inducing 20α-HSD via inhibition of MeCP2 to generate a biologically inactive 20α-OHP.//////////////////MicroRNA-132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1. [Wu S et al. (2015) Estrogen synthesis is an important function of the mammalian ovary. Estrogen plays important roles in many biological processes, including follicular development, oocyte maturation and endometrial proliferation, and dysfunctions in estrogen synthesis contribute to the development of polycystic ovary syndrome and premature ovarian failure. Classical signaling cascades triggered by follicle-stimulating hormone induce estrogen synthesis via the upregulation of Cyp19a1 in granulosa cells (GCs). This study aimed to determine the effect of microRNA-132 (miR-132) on estradiol synthesis in GCs. Primary mouse GCs were collected from ovaries of 21-day-old immature ICR mice through follicle puncture. GCs were cultured and treated with the stable cyclic adenosine monophosphate analog 8-Br-cAMP or transfected with miR-132 mimics, Nurr1-specific small interfering RNA oligonucleotides and Flag-Nurr1 plasmids. Concentrations of estradiol and progesterone in culture medium were determined by an automated chemiluminescence-based assay. Quantitative real time PCR and western blot were performed to identify the effect of miR-132 on Cyp19a1, Cyp11a1 and an orphan nuclear receptor-Nurr1 expression in GCs. Direct suppression of Nurr1 via its 3'-untranslated region by miR-132 were further verified using luciferase reporter assays. The expression level of miR-132 in cultured mouse GCs was significantly elevated during 48 h of treatment with 8-Br-cAMP. The synthesis of estradiol increased after the overexpression of miR-132 in mouse GCs. The real-time PCR results demonstrated that miR-132 induced the expression of Cyp19a1 significantly. Nurr1, an orphan nuclear receptor that suppresses Cyp19a1 expression, was found to be a direct target of miR-132. Nurr1 was suppressed by miR-132, as indicated by a luciferase assay and Western blotting. The knockdown of Nurr1 primarily elevated the synthesis of estradiol and partially attenuated the miR-132-induced estradiol elevation, and the ectopic expression of Flag-Nurr1 abrogated the stimulatory effect of miR-132 on estradiol synthesis in mouse GCs. Our findings suggest that miR-132 is involved in the cAMP signaling pathway and promotes estradiol synthesis via the translational repression of Nurr1 in ovarian GCs.//////////////////
SIRT1 signalling protects mouse oocytes against oxidative stress and is deregulated during aging. Di Emidio G 2014 et al.
STUDY QUESTION
Is SIRT1 involved in the oxidative stress (OS) response in mouse oocytes?
SUMMARY ANSWER
SIRT1 plays a pivotal role in the adaptive response of mouse germinal vesicle (GV) oocytes to OS and promotes a signalling cascade leading to up-regulation of the MnSod gene.
WHAT IS KNOWN ALREADY
OS is known to continuously threaten acquisition and maintenance of oocyte developmental potential during in vivo processes and in vitro manipulations. Previous studies in somatic cells have provided strong evidence for the role of SIRT1 as a sensor of the cell redox state and a protector against OS and aging.
STUDY DESIGN, SIZE, DURATION
GV oocytes obtained from young (4-8 weeks) and reproductively old (48-52 weeks) CD1 mice were blocked in the prophase stage by 0.5 M cilostamide. Groups of 30 oocytes were exposed to 25 M H2O2 and processed following different times for the analysis of intracellular localization of SIRT1 and FOXO3A, and evaluation of Sirt1, miRNA-132, FoxO3a and MnSod gene expression. Another set of oocytes was cultured in the presence or absence of the SIRT1-specific inhibitor Ex527, and exposed to H2O2 in order to assess the involvement of SIRT1 in the activation of a FoxO3a-MnSod axis and ROS detoxification. In the last part of this study, GV oocytes were maturated in vitro in the presence of different Ex527 concentrations (0, 2.5, 5, 10, 20 M) and assessed for maturation rates following 16 h. Effects of Ex527 on spindle morphology and ROS levels were also evaluated.
PARTICIPANTS/MATERIALS, SETTING, METHODS
SIRT1 and FOXO3A intracellular distribution in response to OS was investigated by immunocytochemistry. Real-time RT-PCR was employed to analyse Sirt1, miR-132, FoxO3a and MnSod gene expression. Reactive oxygen species (ROS) production was evaluated by in vivo measurement of carboxy-H2DCF diacetate labelling. Spindle and chromosomal distribution in in vitro matured oocytes were analysed by immunocytochemistry and DNA fluorescent labelling, respectively.
MAIN RESULTS AND THE ROLE OF CHANCE
Specific changes in the intracellular localization of SIRT1 and up-regulation of Sirt1 gene were detected in mouse oocytes in response to OS. Moreover, increased intracellular ROS were observed when SIRT1 activity was inhibited by Ex527. In aged oocytes Sirt1 was expressed more than in young oocytes but SIRT1 protein was undetectable. Upon OS, significant changes in miR-132 micro-RNA, a validated Sirt1 modulator, were observed. A negative correlation between Sirt1 mRNA and miR-132 levels was observed when young oocytes exposed to OS were compared with young control oocytes, and when aged oocytes were compared with young control oocytes. FoxO3a and MnSod transcripts were increased upon OS with the same kinetics as Sirt1 transcripts, and up-regulation of MnSod gene was prevented by oocyte treatment with Ex527, indicating that SIRT1 acts upstream to the FoxO3a-MnSod axis. Finally, the results of the in vitro maturation assay suggested that SIRT1 might be involved in oocyte maturation by regulating the redox state and ensuring normal spindle assembly.
LIMITATIONS, REASONS FOR CAUTION
The main limitation of this study was the absence of direct quantification of SIRT1 enzymatic activity due to the lack of an appropriately sensitive method.
WIDER IMPLICATIONS OF THE FINDINGS
The present findings may provide a valuable background for studying the regulation of SIRT1 during oogenesis and its relevance as a sensor of oocyte redox state and energy status. The antioxidant response orchestrated by SIRT1 in oocytes seems to decrease with aging. This suggests that SIRT1 could be an excellent pharmacological target for improving oocyte quality and IVF outcome in aging or aging-like diseases.
STUDY FUNDING/COMPETING INTERESTS
The work was supported by the Ministero dell'Universit e della Ricerca Scientifica (MIUR) to C.T., F.A., C.D., A.M.D. The authors declare no conflict of interest.
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Expression regulated by
LH
Comment
Ovarian localization
Oocyte, Granulosa, Follicular Fluid
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Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo. Sang Q et al. Context:Human follicular fluid is a combination of proteins, metabolites, and ionic compounds that is indicative of the general state of follicular metabolism and is associated with maturation and quality of oocytes. Deviations in these components are often associated with reproductive diseases. There has been no report of microRNAs (miRNAs) in human follicular fluids.Objective:We hypothesized that human follicular fluid may contain miRNAs. We sought to identify cell-free miRNAs in human follicular fluid and to investigate these miRNAs's function in vitro and any roles they play in polycystic ovary syndrome.Design:Genome-wide deep sequencing and TaqMan miRNA arrays were used to identify miRNAs, and the roles of the highly expressed miRNAs in steroidogenesis were investigated in KGN cells. Quantification of candidate miRNAs in follicular fluids of PCOS and controls was performed using TaqMan miRNA assays.Results:We identified miRNAs in microvesicles and the supernatant of human follicular fluid. Bioinformatics analysis showed that the most highly expressed miRNAs targeted genes associated with reproductive, endocrine, and metabolic processes. We found that miR-132, miR-320, miR-520c-3p, miR-24, and miR-222 regulate estradiol concentrations and that miR-24, miR-193b, and miR-483-5p regulate progesterone concentrations. Finally, we showed that miR-132 and miR-320 are expressed at significantly lower levels in the follicular fluid of polycystic ovary patients than in healthy controls (p =0.005, p =0.0098, respectively)Conclusion:These results demonstrate that there are numerous miRNAs in human follicular fluids, some of which play important roles in steroidogenesis and PCOS. This study substantially revises our understanding of the content of human follicular fluid and lays the foundation for the future investigation of miRNAs' role in PCOS.
Hormonal Regulation of MicroRNA Expression in Periovulatory Mouse Mural Granulosa Cells. Fiedler SD et al. MicroRNAs (miRNA) mediate post-transcriptional gene regulation by binding to the 3'untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory surge of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG-induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212, were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect post-transcriptional gene regulation within these cells.
Follicle stages
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Quantitative RT-PCR Methods for Mature microRNA Expression Analysis. Fiedler SD et al. This chapter describes two methods to measure expression of mature miRNA levels using qRT-PCR. The first method uses stem-loop RT primers to produce cDNA for specific miRNAs, a technique that our laboratory has modified to increase the number of miRNAs being reverse transcribed within a single RT reaction from one (as suggested by the manufacturer) to five. The second method uses a modified oligo(dT) technique to reverse transcribe all transcripts within an RNA sample; therefore, target miRNA and normalizing mRNA can be analyzed from the same RT reaction. We examined the level of miRNA-132, a miRNA known to be upregulated in granulosa cells following hCG treatment, using both of these methods. Data were normalized to GAPDH or snU6 and evaluated by DeltaDeltaCt and standard curve analysis. There was no significant difference (P > 0.05) in miRNA-132 expression between the stem-loop and modified oligo(dT) RT methods indicating that both are statistically equivalent. However, from a technical point of view, the modified oligo(dT) method was less time consuming and required only a single RT reaction to reverse transcribe both miRNA and mRNA.