Competing endogenous RNA network mediated by circ_3205 in SARS-CoV-2 infected cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new member of the Betacoronaviridae family, responsible for the recent pandemic outbreak of COVID-19. To start exploring the molecular events that follow host cell infection, we queried VirusCircBase and identified a circular RNA (circRNA) predicted to be synthesized by SARS-CoV-2, circ_3205, which we used to probe: (i) a training cohort comprised of two pools of cells from three nasopharyngeal swabs of SARS-CoV-2 infected (positive) or uninfected (negative, UCs) individuals; (ii) a validation cohort made up of 12 positive and 3 negative samples. The expression of circRNAs, miRNAs and miRNA targets was assayed through real-time PCR. CircRNA-miRNA interactions were predicted by TarpMiR, Analysis of Common Targets for circular RNAs (ACT), and STarMir tools. Enrichment of the biological processes and the list of predicted miRNA targets were retrieved from DIANA miRPath v3.0. Our results showed that the predicted SARS-CoV-2 circ_3205 was expressed only in positive samples and its amount positively correlated with that of SARS-CoV-2 Spike (S) mRNA and the viral load (r values = 0.80952 and 0.84867, Spearman's correlation test, respectively). Human (hsa) miR-298 was predicted to interact with circ_3205 by all three predictive tools. KCNMB4 and PRKCE were predicted as hsa-miR-298 targets. Interestingly, the function of both is correlated with blood coagulation and immune response. KCNMB4 and PRKCE mRNAs were upregulated in positive samples as compared to UCs (6 and 8.1-fold, p values = 0.049 and 0.02, Student's t test, respectively) and their expression positively correlated with that of circ_3205 (r values = 0.6 and 0.25, Spearman's correlation test, respectively). We propose that our results convincingly suggest that circ_3205 is a circRNA synthesized by SARS-CoV-2 upon host cell infection and that it may behave as a competitive endogenous RNA (ceRNA), sponging hsa-miR-298 and contributing to the upregulation of KCNMB4 and PRKCE mRNAs.

Down-regulation of long non-coding RNAs in reproductive aging and analysis of the lncRNA-miRNA-mRNA networks in human cumulus cells.

PURPOSE: Long non-coding RNAs (lncRNAs) control gene expression at multiple levels. By interacting with microRNAs (miRNAs), they regulate their mRNA targets creating dynamic regulatory networks involved in different cellular processes. Their role in follicle development and oocyte maturation has recently emerged. lncRNA deregulation has been found associated with different pathological conditions. In this study, we identified differentially expressed lncRNAs in cumulus cells (CCs) isolated from MII oocytes of advanced maternal age women and proposed ceRNA-networks involved in signaling pathways crucial in ovarian folliculogenesis and female germ cell maturation. METHODS: We performed a high-throughput analysis of the expression profile of 68 lncRNAs from CCs of aged and young women by using NanoString technology. By miRNet, TarPmiR, miRTarBase, OKdb, and KEGG we predicted some ceRNA-networks involving the differentially expressed (DE) lncRNAs, miRNA interactors, and their mRNA target genes. RESULTS: We identified 28 lncRNAs down-regulated in CC samples from aged women. The analysis revealed that the miRNAs binding 11 of the DE lncRNAs and their mRNA targets are included in ceRNA-networks involved in the regulation of the PI3K-Akt, FOXO, and p53 signaling pathways. CONCLUSION: We proposed that the lncRNA down-regulation in CCs from aged women could influence the expression of genes encoding proteins deregulated in reproductive aging. A better understanding of the interplay of lncRNA-miRNA-mRNA networks in human CCs could increase our knowledge about the mechanisms of regulation of gene expression involved in aging, lead to the development of novel therapeutics, and improve reproductive outcomes in aged women.

circSMARCA5 Is an Upstream Regulator of the Expression of miR-126-3p, miR-515-5p, and Their mRNA Targets, Insulin-like Growth Factor Binding Protein 2 (IGFBP2) and NRAS Proto-Oncogene, GTPase (NRAS) in Glioblastoma.

The involvement of non-coding RNAs (ncRNAs) in glioblastoma multiforme (GBM) pathogenesis and progression has been ascertained but their cross-talk within GBM cells remains elusive. We previously demonstrated the role of circSMARCA5 as a tumor suppressor (TS) in GBM. In this paper, we explore the involvement of circSMARCA5 in the control of microRNA (miRNA) expression in GBM. By using TaqMan® low-density arrays, the expression of 748 miRNAs was assayed in U87MG overexpressing circSMARCA5. Differentially expressed (DE) miRNAs were validated through single TaqMan® assays in: (i) U87MG overexpressing circSMARCA5; (ii) four additional GBM cell lines (A172; CAS-1; SNB-19; U251MG); (iii) thirty-eight GBM biopsies; (iv) twenty biopsies of unaffected brain parenchyma (UC). Validated targets of DE miRNAs were selected from the databases TarBase and miRTarbase, and the literature; their expression was inferred from the GBM TCGA dataset. Expression was assayed in U87MG overexpressing circSMARCA5, GBM cell lines, and biopsies through real-time PCR. TS miRNAs 126-3p and 515-5p were upregulated following circSMARCA5 overexpression in U87MG and their expression was positively correlated with that of circSMARCA5 (r-values = 0.49 and 0.50, p-values = 9 × 10-5 and 7 × 10-5, respectively) in GBM biopsies. Among targets, IGFBP2 (target of miR-126-3p) and NRAS (target of miR-515-5p) mRNAs were positively correlated (r-value = 0.46, p-value = 0.00027), while their expression was negatively correlated with that of circSMARCA5 (r-values = -0.58 and -0.30, p-values = 0 and 0.019, respectively), miR-126-3p (r-value = -0.36, p-value = 0.0066), and miR-515-5p (r-value = -0.34, p-value = 0.010), respectively. Our data identified a new GBM subnetwork controlled by circSMARCA5, which regulates downstream miRNAs 126-3p and 515-5p, and their mRNA targets IGFBP2 and NRAS.

Molecular profiling of follicular fluid microRNAs in young women affected by Hodgkin lymphoma.

RESEARCH QUESTION: Treatments for Hodgkin lymphoma have improved but one of their common effects is gonadal toxicity, which contributes to fertility damage of patients and induces temporary or irreversible loss of fertility. Could micro-RNA (miRNA) expression profiles in follicular fluid be influenced by Hodgkin lymphoma? Could their alteration affect molecular pathways involved in follicle growth and oocyte maturation? DESIGN: miRNA expression profile was investigated in follicular fluid samples from young women affected by Hodgkin lymphoma compared with healthy controls by NanoString technology. Bioinformatic analysis was used to verify miRNA involvement in follicle development and miRNA deregulation with Hodgkin lymphoma in a larger cohort of follicular fluid samples was confirmed by real-time quantitative polymerase chain reaction. RESULTS: Thirteen miRNAs are deregulated in Hodgkin lymphoma samples compared with controls and are involved in molecular pathways related to cancer, gametogenesis and embryogenesis. Among them, let-7b-5p, miR-423-5p, miR-503-5p, miR-574-5p and miR-1303 are implicated in biological processes related to follicle development and oocyte maturation. Let-7b-5p holds the central position in the regulatory network of miRNA-mRNA interactions, has the highest number of mRNA target genes shared with the other differentially expressed miRNAs and is significantly downregulated in Hodgkin lymphoma follicular fluid samples. CONCLUSIONS: These data led us to question the potential influence of miRNA deregulation on oocyte quality. Further studies are needed to verify the reproductive potential of young patients with Hodgkin lymphoma before starting chemotherapy protocols and an adequate protocol of fertility preservation needs to be guaranteed.

MicroRNA-Mediated Regulation of the Virus Cycle and Pathogenesis in the SARS-CoV-2 Disease.

In the last few years, microRNA-mediated regulation has been shown to be important in viral infections. In fact, viral microRNAs can alter cell physiology and act on the immune system; moreover, cellular microRNAs can regulate the virus cycle, influencing positively or negatively viral replication. Accordingly, microRNAs can represent diagnostic and prognostic biomarkers of infectious processes and a promising approach for designing targeted therapies. In the past 18 months, the COVID-19 infection from SARS-CoV-2 has engaged many researchers in the search for diagnostic and prognostic markers and the development of therapies. Although some research suggests that the SARS-CoV-2 genome can produce microRNAs and that host microRNAs may be involved in the cellular response to the virus, to date, not enough evidence has been provided. In this paper, using a focused bioinformatic approach exploring the SARS-CoV-2 genome, we propose that SARS-CoV-2 is able to produce microRNAs sharing a strong sequence homology with the human ones and also that human microRNAs may target viral RNA regulating the virus life cycle inside human cells. Interestingly, all viral miRNA sequences and some human miRNA target sites are conserved in more recent SARS-CoV-2 variants of concern (VOCs). Even if experimental evidence will be needed, in silico analysis represents a valuable source of information useful to understand the sophisticated molecular mechanisms of disease and to sustain biomedical applications.

The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential.

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the "Find Individual Motif Occurrences" (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.

IUI and uterine lavage of in vivo-produced blastocysts for PGT purposes: is it a technically and ethically reasonable perspective? Is it actually needed?

A recent study by Munné et al. portrayed a protocol to retrieve in vivo produced blastocysts after IUI and uterine lavage for preimplantation genetic testing (PGT) purposes. The authors claimed this protocol might represent a reasonable future perspective for patients who do not want to undergo IVF, but still want to be informed about their embryos' genetic/chromosomal defects. Although the intent of making PGT available also to patients who cannot or do not need to undergo IVF is respectable, the value of this study is undermined by severe technical and ethical issues. Munné and colleagues' paper was discussed within the executive committee (i.e., president and vice-president of the society, director and vice-director of the scientific committee, secretariat, and counselors), the special interest group in reproductive genetics, the scientific committee, and the collegio dei probiviri of the Italian Society of Embryology, Reproduction and Research (SIERR). The points raised from this discussion are summarized in this opinion paper.

Uncharacterized RNAs in Plasma of Alzheimer's Patients Are Associated with Cognitive Impairment and Show a Potential Diagnostic Power.

Alzheimer's disease (AD) diagnosis is actually based on clinical evaluation and brain-imaging tests, and it can often be confirmed only post-mortem. Therefore, new non-invasive molecular biomarkers are necessary to improve AD diagnosis. As circulating microRNA biomarkers have been proposed for many diseases, including AD, we aimed to identify new diagnostic non-small RNAs in AD. Whole transcriptome analysis was performed on plasma samples of five AD and five unaffected individuals (CTRL) using the Clariom D Pico Assay, followed by validation in real-time PCR on 37 AD patients and 37 CTRL. Six differentially expressed (DE) transcripts were identified: GS1-304P7.3 (upregulated), NONHSAT090268, TC0100011037, TC0400008478, TC1400008125, and UBE2V1 (downregulated). Peripheral blood mononuclear cells (PBMCs) may influence the expression of circulating RNAs and their analysis has been proposed to improve AD clinical management. Accordingly, DE transcript expression was also evaluated in PBMCs, showing no difference between AD and CTRL. ROC (receiver operating characteristic) curve analysis was performed to evaluate the diagnostic accuracy of each DE transcript and a signature including all of them. A correlation between cognitive impairment and GS1-304P7.3, NONHSAT090268, TC0100011037, and TC0400008478 was detected, suggesting a potential association between their extracellular abundance and AD clinical phenotype. Finally, this study identified six transcripts showing altered expression in the plasma of AD patients. Given the need for new, accurate blood biomarkers for AD diagnosis, these transcripts may be considered for further analyses in larger cohorts, also in combination with other biomarkers, aiming to identify specific RNA-based biomarkers to be eventually applied to clinical practice.

Potential Associations Among Alteration of Salivary miRNAs, Saliva Microbiome Structure, and Cognitive Impairments in Autistic Children.

Recent evidence has demonstrated that salivary molecules, as well as bacterial populations, can be perturbed by several pathological conditions, including neuro-psychiatric diseases. This relationship between brain functionality and saliva composition could be exploited to unveil new pathological mechanisms of elusive diseases, such as Autistic Spectrum Disorder (ASD). We performed a combined approach of miRNA expression profiling by NanoString technology, followed by validation experiments in qPCR, and 16S rRNA microbiome analysis on saliva from 53 ASD and 27 neurologically unaffected control (NUC) children. MiR-29a-3p and miR-141-3p were upregulated, while miR-16-5p, let-7b-5p, and miR-451a were downregulated in ASD compared to NUCs. Microbiome analysis on the same subjects revealed that Rothia, Filifactor, Actinobacillus, Weeksellaceae, Ralstonia, Pasteurellaceae, and Aggregatibacter increased their abundance in ASD patients, while Tannerella, Moryella and TM7-3 decreased. Variations of both miRNAs and microbes were statistically associated to different neuropsychological scores related to anomalies in social interaction and communication. Among miRNA/bacteria associations, the most relevant was the negative correlation between salivary miR-141-3p expression and Tannerella abundance. MiRNA and microbiome dysregulations found in the saliva of ASD children are potentially associated with cognitive impairments of the subjects. Furthermore, a potential cross-talking between circulating miRNAs and resident bacteria could occur in saliva of ASD.

Upregulated microRNAs in membranous glomerulonephropathy are associated with significant downregulation of IL6 and MYC mRNAs.

Membranous glomerulonephropathy (MGN) is a glomerulopathy characterized by subepithelial deposits of immune complexes on the extracapillary side of the glomerular basement membrane. Insertion of C5b-9 (complement membrane-attack complex) into the membrane leads to functional impairment of the glomerular capillary wall. Knowledge of the molecular pathogenesis of MGN is actually scanty. MicroRNA (miRNA) profiling in MGN and unaffected tissues was performed by TaqMan Low-Density Arrays. Expression of miRNAs and miRNA targets was evaluated in Real-Time polymerase chain reaction (PCR). In vitro transient silencing of miRNAs was achieved through transfection with miRNA inhibitors. Ten miRNAs (let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, miR-107, miR-129-3p, miR-423-5p, miR-516-3p, miR-532-3p, and miR-1275) were differentially expressed (DE) in MGN biopsies compared to unaffected controls. Interleukin 6 (IL6) and MYC messenger RNAs (mRNAs; targets of DE miRNAs) were significantly downregulated in biopsies from MGN patients, and upregulated in A498 cells following let-7a-5p or let-7c-5p transient silencing. Gene ontology analysis showed that DE miRNAs regulate pathways associated with MGN pathogenesis, including cell cycle, proliferation, and apoptosis. A significant correlation between DE miRNAs and mRNAs and clinical parameters (i.e., antiphospholipid antibodies, serum creatinine, estimated glomerular filtration, proteinuria, and serum cholesterol) has been detected. Based on our data, we propose that DE miRNAs and their downstream network may be involved in MGN pathogenesis and could be considered as potential diagnostic biomarkers of MGN.

CircNAPEPLD is expressed in human and murine spermatozoa and physically interacts with oocyte miRNAs.

Circular RNAs (circRNAs) have a critical role in the control of gene expression. Their function in spermatozoa (SPZ) is unknown to date. Twenty-eight genes, involved in SPZ/testicular and epididymal physiology, were given in circBase database to find which of them may generate circular transcripts. We focused on circNAPEPLDiso1, one of the circular RNA isoforms of NAPEPLD transcript, because expressed in human and murine SPZ. In order to functionally characterize circNAPEPLDiso1 as potential microRNA (miRNA) sponge, we performed circNAPEPLDiso1-miR-CATCH and then profiled the expression of 754 miRNAs, by using TaqMan® Low Density Arrays. Among them, miRNAs 146a-5p, 203a-3p, 302c-3p, 766-3p and 1260a (some of them previously shown to be expressed in the oocyte), resulted enriched in circNAPEPLDiso1-miR-CATCHed cell lysate: the network of interactions generated from their validated targets was centred on a core of genes involved in the control of cell cycle. Moreover, computational analysis of circNAPEPLDiso1 sequence also showed its potential translation in a short form of NAPEPLD protein. Interestingly, the expression analysis in murine-unfertilized oocytes revealed low and high levels of circNAPEPLDiso1 and circNAPEPLDiso2, respectively. After fertilization, circNAPEPLDiso1 expression significantly increased, instead circNAPEPLDiso2 expression appeared constant. Based on these data, we suggest that SPZ-derived circNAPEPLDiso1 physically interacts with miRNAs primarily involved in the control of cell cycle; we hypothesize that it may represent a paternal cytoplasmic contribution to the zygote and function as a miRNA decoy inside the fertilized oocytes to regulate the first stages of embryo development. This role is proposed here for the first time.

Extracellular Vesicles in Human Oogenesis and Implantation.

Reproduction, the ability to generate offspring, represents one of the most important biological processes, being essential for the conservation of the species. In mammals, it involves different cell types, tissues and organs, which, by several signaling molecules, coordinate the different events such as gametogenesis, fertilization and embryo development. In the last few years, the role of Extracellular Vesicles, as mediators of cell communication, has been investigated in every phase of these complex processes. Microvesicles and exosomes, identified in the fluid of ovarian follicles during egg maturation, are involved in communication between the developing oocyte and the somatic follicular cells. More recently, it has been demonstrated that, during implantation, Extracellular Vesicles could participate in the complex dialog between the embryo and maternal tissues. In this review, we will focus our attention on extracellular vesicles and their cargo in human female reproduction, mainly underlining the involvement of microRNAs in intercellular communication during the several phases of the reproductive process.

CircSMARCA5 Inhibits Migration of Glioblastoma Multiforme Cells by Regulating a Molecular Axis Involving Splicing Factors SRSF1/SRSF3/PTB.

Circular RNAs (circRNAs) have recently emerged as a new class of RNAs, highly enriched in the brain and very stable within cells, exosomes and body fluids. To analyze their involvement in glioblastoma multiforme (GBM) pathogenesis, we assayed the expression of twelve circRNAs, physiologically enriched in several regions of the brain, through real-time PCR in a cohort of fifty-six GBM patient biopsies and seven normal brain parenchymas. We focused on hsa_circ_0001445 (circSMARCA5): it was significantly downregulated in GBM biopsies as compared to normal brain tissues (p-value < 0.00001, student's t-test), contrary to its linear isoform counterpart that did not show any differential expression (p-value = 0.694, student's t-test). Analysis of a public dataset revealed a negative correlation between the expression of circSMARCA5 and glioma's histological grade, suggesting its potential negative role in the progression to malignancy. Overexpressing circSMARCA5 in U87MG cells significantly decreased their migration, but not their proliferation rate. In silico scanning of circSMARCA5 sequence revealed an enrichment in binding motifs for several RNA binding proteins (RBPs), specifically involved in splicing. Among them, serine and arginine rich splicing factor 1 (SRSF1), a splicing factor known to be a positive controller of cell migration and known to be overexpressed in GBM, was predicted to bind circSMARCA5 by three different prediction tools. Direct interaction between circSMARCA5 and SRSF1 is supported by enhanced UV crosslinking and immunoprecipitation (eCLIP) data for SRSF1 in K562 cells from Encyclopedia of DNA Elements (ENCODE). Consistently, U87MG overexpressing circSMARCA5 showed an increased expression of serine and arginine rich splicing factor 3 (SRSF3) RNA isoform containing exon 4, normally skipped in a SRSF1-dependent manner, resulting in a non-productive non-sense mediated decay (NMD) substrate. Interestingly, SRSF3 is known to interplay with two other splicing factors, polypyrimidine tract binding protein 1 (PTBP1) and polypyrimidine tract binding protein 2 (PTBP2), that positively regulate glioma cells migration. Collectively, our data show circSMARCA5 as a promising druggable tumor suppressor in GBM and suggest that it may exert its function by tethering the RBP SRSF1.

Epigenetic dysregulation in neuroblastoma: A tale of miRNAs and DNA methylation.

In neuroblastoma, the epigenetic landscape is more profoundly altered in aggressive compared to lower grade tumors and the concomitant hypermethylation of many genes, defined as "methylator phenotype", has been associated with poor outcome. DNA methylation can interfere with gene expression acting at distance through the methylation or demethylation of the regulatory regions of miRNAs. The multiplicity of miRNA targets may result in the simultaneous alteration of many biological pathways like cell proliferation, apoptosis, migration and differentiation. We have analyzed the methylation status of a set of miRNAs in a panel of neuroblastoma cell lines and identified a subset of hypermethylated and down-regulated miRNAs (miRNA 34b-3p, miRNA 34b-5p, miRNA34c-5p, and miRNA 124-2-3p) involved in the regulation of cell cycle, apoptosis and in the control of MYCN expression. These miRNAs share, in part, some of the targets whose expression is inversely correlated to the methylation and expression of the corresponding miRNA. To simulate the effect of the demethylation of miRNAs, we transfected the corresponding miRNA-mimics in the same cell lines and observed the down-regulation of a set of their target genes as well as the partial block of the cell cycle and the activation of the apoptotic pathway. The epigenetic alterations of miRNAs described in the present study were found also in a subset of patients at high risk of progression. Our data disclosed a complex network of interactions between epigenetically altered miRNAs and target genes, that could interfere at multiple levels in the control of cell homeostasis.

MicroRNAs Are Stored in Human MII Oocyte and Their Expression Profile Changes in Reproductive Aging.

Maternal RNAs are synthesized by the oocyte during its growth; some of them are utilized for oocyte-specific processes and metabolism, others are stored and used during early development before embryonic genome activation. The appropriate expression of complex sets of genes is needed for oocyte maturation and early embryo development. In spite of the basic role of noncoding RNAs in the regulation of gene expression, few studies have analyzed their role in human oocytes. In this study, we identified the microRNAs (miRNAs) expressed in human metaphase II stage oocytes, and found that some of them are able to control pluripotency, chromatin remodeling, and early embryo development. We demonstrated that 12 miRNAs are differentially expressed in women of advanced reproductive age and, by bioinformatics analysis, we identified their mRNA targets, expressed in human oocytes and involved in the regulation of pathways altered in reproductive aging. Finally, we found the upregulation of miR-29a-3p, miR-203a-3p, and miR-494-3p, evolutionarily conserved miRNAs, also in aged mouse oocytes, and demonstrated that their overexpression is antithetically correlated with the downregulation of DNA methyltransferase 3A (Dnmt3a), DNA methyltransferase 3B (Dnmt3b), phosphatase and tensin homolog (Pten), and mitochondrial transcription factor A (Tfam). We propose that oocyte miRNAs perform an important regulatory function in human female germ cells, and their altered regulation could explain the changes occurring in oocyte aging.

SIRT1 signalling protects mouse oocytes against oxidative stress and is deregulated during aging.

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 µM cilostamide. Groups of 30 oocytes were exposed to 25 µM 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 µM) 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'Università e della Ricerca Scientifica (MIUR) to C.T., F.A., C.D., A.M.D. The authors declare no conflict of interest.

A Circular RNA Derived from the Pumilio 1 Gene Could Regulate PTEN in Human Cumulus Cells.

CircRNAs are a class of non-coding RNAs able to regulate gene expression at multiple levels. Their involvement in physiological processes, as well as their altered regulation in different human diseases, both tumoral and non-tumoral, is well documented. However, little is known about their involvement in female reproduction. This study aims to identify circRNAs potentially involved in reproductive women's health. Candidate circRNAs expressed in ovary and sponging miRNAs, already known to be expressed in the ovary, were selected by a computational approach. Using real time PCR, we verified their expression and identified circPUM1 as the most interesting candidate circRNA for further analyses. We assessed the expression of circPUM1 and its linear counterpart in all the follicle compartments and, using a computational and experimental approach, identified circPUM1 direct and indirect targets, miRNAs and mRNAs, respectively, in cumulus cells. We found that both circPUM1 and its mRNA host gene are co-expressed in all the follicle compartments and proposed circPUM1 as a potential regulator of PTEN, finding a strong positive correlation between circPUM1 and PTEN mRNA. These results suggest a possible regulation of PTEN by circPUM1 in cumulus cells and point out the important role of circRNA inside the pathways related to follicle growth and oocyte maturation.

miR-296-3p, miR-298-5p and their downstream networks are causally involved in the higher resistance of mammalian pancreatic α cells to cytokine-induced apoptosis as compared to ß cells.

BACKGROUND: The molecular bases of mammalian pancreatic α cells higher resistance than ß to proinflammatory cytokines are very poorly defined. MicroRNAs are master regulators of cell networks, but only scanty data are available on their transcriptome in these cells and its alterations in diabetes mellitus. RESULTS: Through high-throughput real-time PCR, we analyzed the steady state microRNA transcriptome of murine pancreatic α (αTC1-6) and ß (ßTC1) cells: their comparison demonstrated significant differences. We also characterized the alterations of αTC1-6 cells microRNA transcriptome after treatment with proinflammatory cytokines. We focused our study on two microRNAs, miR-296-3p and miR-298-5p, which were: (1) specifically expressed at steady state in αTC1-6, but not in ßTC1 or INS-1 cells; (2) significantly downregulated in αTC1-6 cells after treatment with cytokines in comparison to untreated controls. These microRNAs share more targets than expected by chance and were co-expressed in αTC1-6 during a 6-48 h time course treatment with cytokines. The genes encoding them are physically clustered in the murine and human genome. By exploiting specific microRNA mimics, we demonstrated that experimental upregulation of miR-296-3p and miR-298-5p raised the propensity to apoptosis of transfected and cytokine-treated αTC1-6 cells with respect to αTC1-6 cells, treated with cytokines after transfection with scramble molecules. Both microRNAs control the expression of IGF1Rß, its downstream targets phospho-IRS-1 and phospho-ERK, and TNFα. Our computational analysis suggests that MAFB (a transcription factor exclusively expressed in pancreatic α cells within adult rodent islets of Langerhans) controls the expression of miR-296-3p and miR-298-5p. CONCLUSIONS: Altogether, high-throughput microRNA profiling, functional analysis with synthetic mimics and molecular characterization of modulated pathways strongly suggest that specific downregulation of miR-296-3p and miR-298-5p, coupled to upregulation of their targets as IGF1Rß and TNFα, is a major determinant of mammalian pancreatic α cells resistance to apoptosis induction by cytokines.

The apoptotic transcriptome of the human MII oocyte: characterization and age-related changes.

Fully competent oocytes represent the final outcome of a highly selective process. The decline of oocyte competence with ageing, coupled to quantitative decrease of ovarian follicles has been well established; on the contrary, its molecular bases are still poorly understood. Through quantitative high throughput PCR, we investigated the role of apoptotic machinery (AM) in this process. To this aim, we determined AM transcriptome in mature MII oocyte pools from women aged more than 38 years (cohort A), and compared to women aged up to 35 years (cohort B). Subsequently, 10 representative AM genes were selected and analyzed in 33 single oocytes (15 from cohort A and 18 from cohort B). These investigations led us to identify: (1) the significant upregulation of proapoptotic genes such us CD40, TNFRSF10A, TNFRSF21 and the downregulation of antiapoptotic genes such as BCL2 and CFLAR in cohort A respect to cohort B; (2) AM transcripts that have not previously been reported in human oocytes (BAG3, CD40, CFLAR, TNFRSF21, TRAF2, TRAF3). Our results demonstrated that during maturation the oocytes from older women selectively accumulate mRNAs that are able to trigger the extrinsic apoptotic pathway. These data contribute to clarify the molecular mechanisms of AM involvement in the natural selection strategy of removing low quality oocytes and preventing unfit or poorly fit embryos.

MicroRNAs in vitreus humor from patients with ocular diseases.

PURPOSE: Based on evidence that microRNAs (miRNAs) are found in many biologic fluids (e.g., urine, saliva, pleural fluid), we sought to detect the presence of miRNAs and analyze their profile in vitreous humor (VH) from patients affected by various ocular diseases. METHODS: MiRNAs were purified from VH samples taken during vitrectomy, by using the Qiagen miRNeasy Mini Kit. The expression profile on 745 miRNAs was performed by using TaqMan Low Density Array. Single TaqMan expression assays were performed on 18 VH samples (six each from patients with choroidal melanomas, retinal detachment, or macular hole) for miRNAs commonly expressed in serum or retinal cells: let-7b, miR-21, miR-26a, miR-146a, miR-199-3p, miR-210, miR-374a*, miR-532-5p. RNA extracted from serum of six healthy donors or from formalin-fixed, paraffin-embedded samples of choroidal melanocytes from four uveal melanomas (epithelioid cells) and from three unaffected eyes were used as controls. RESULTS: We identified the presence of 94 circulating small RNAs in the vitreous, some of which (miR-9, miR-9*, miR-125a-3p, miR-184, miR-211, miR-214, miR-302c, miR-452, miR-628, miR-639) are particularly abundant in the VH but downrepresented or not detectable in serum. MiR-146a and miR-26a were overexpressed more than threefold in VH from patients with uveal melanomas compared to the other pathological groups (Wilcoxon signed-rank test, p value < 0.05). CONCLUSIONS: Our experimental data suggest that a specific set of circulating miRNAs is secreted in the vitreous, which is quite different from the miRNA pattern in serum, and that the quantity of vitreal miRNAs could change, depending on the pathologies of the eye.