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RNAs, especially non-coding RNAs (ncRNAs), are crucial players in regulating cellular mechanisms due to their ability to interact with and regulate other molecules. Altered expression patterns of ncRNAs have been observed in prostate cancer (PCa), contributing to the disease's initiation, progression, and treatment response. This study aimed to evaluate the ability of a specific set of RNAs, including long ncRNAs (lncRNAs), microRNAs (miRNAs), and mRNAs, to discriminate between PCa and the non-neoplastic condition benign prostatic hyperplasia (BPH). After selecting by literature mining the most relevant RNAs differentially expressed in biofluids from PCa patients, we evaluated their discriminatory power in samples of unfiltered urine from 50 PCa and 50 BPH patients using both real-time PCR and droplet digital PCR (ddPCR). Additionally, we also optimized a protocol for urine sample manipulation and RNA extraction. This two-way validation study allowed us to establish that miRNAs (i.e., miR-27b-3p, miR-574-3p, miR-30a-5p, and miR-125b-5p) are more efficient biomarkers for PCa compared to long RNAs (mRNAs and lncRNAs) (e.g., PCA3, PCAT18, and KLK3), as their dysregulation was consistently reported in the whole urine of patients with PCa compared to those with BPH in a statistically significant manner regardless of the quantification methodology performed. Moreover, a significant increase in diagnostic performance was observed when molecular signatures composed of different miRNAs were considered. Hence, the abovementioned circulating ncRNAs represent excellent potential non-invasive biomarkers in urine capable of effectively distinguishing individuals with PCa from those with BPH, potentially reducing cancer overdiagnosis.

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.

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 .

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Ovarian aging affects female reproductive potential and is characterized by alterations in proteins, mRNAs and non-coding RNAs inside the ovarian follicle. Ovarian somatic cells and the oocyte communicate with each other secreting different molecules into the follicular fluid, by extracellular vesicles. The cargo of follicular fluid vesicles may influence female reproductive ability; accordingly, analysis of extracellular vesicle content could provide information about the quality of the female germ cell.In order to identify the most significant deregulated microRNAs in reproductive aging, we quantified the small extracellular vesicles in human follicular fluid from older and younger women and analyzed the expression of microRNAs enclosed inside the vesicles. We found twice as many small extracellular vesicles in the follicular fluid from older women and several differentially expressed microRNAs. Correlating microRNA expression profiles with vesicle number, we selected 46 deregulated microRNAs associated with aging. Bioinformatic analyses allowed us to identify six miRNAs involved in TP53 signaling pathways. Specifically, miR-16-5p, miR214-3p and miR-449a were downregulated and miR-125b, miR-155-5p and miR-372 were upregulated, influencing vesicle release, oocyte maturation and stress response. We believe that this approach allowed us to identify a battery of microRNAs strictly related to female reproductive aging.

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.

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., (© 2018 Wiley Periodicals, Inc.)

In this study, for the first time, we demonstrated the presence of microRNAs and extracellular vesicles in human blastocoel fluid. The bioinformatic and comparative analyses identified the biological function of blastocoel fluid microRNAs and suggested a potential role inside the human blastocyst. We found 89 microRNAs, expressed at different levels, able to regulate critical signaling pathways controlling embryo development, such as pluripotency, cell reprogramming, epigenetic modifications, intercellular communication, cell adhesion and cell fate. Blastocoel fluid microRNAs reflect the miRNome of embryonic cells and their presence, associated with the discovery of extracellular vesicles, inside blastocoel fluid, strongly suggests their important role in mediating cell communication among blastocyst cells. Their characterization is important to better understand the earliest stages of embryogenesis and the complex circuits regulating pluripotency. Moreover, blastocoel fluid microRNA profiles could be influenced by blastocyst quality, therefore, microRNAs might be used to assess embryo potential in IVF cycles.

Problem: Chronic endometritis (CE) is usually asymptomatic and different studies demonstrated the relation with infertility and recurrent pregnancy loss. Altered regulation of protein-encoding genes in CE has been demonstrated, but no evidence about the involvement of microRNAs in the pathology is present in literature., Method of Study: In the endometrium from 15 women with CE and 15 healthy women, by RT-qPCR single assays, we investigated some microRNAs targeting IL11, CCL4, IGF1, and IGFBP1, which mRNAs had been found differentially expressed in endometrium of women affected by CE. The expression of IGF1 and IL11, targets of the deregulated microRNAs, has been analyzed in the same endometrium samples. We assessed the expression profiles of the deregulated microRNAs in the serum of the same patients validating their ability as biomarkers by statistical analysis., Results: We demonstrated the upregulation of miR-27a-3p and miR-124-3p in the endometrium and serum from women with CE and found an anticorrelation relationship between miR-27a-3p and IGF1 in endometrium. ROC curve analysis suggested that miRNA investigation in endometrium and serum could discriminate women with CE., Conclusion: MiR-27a-3p and miR-124-3p could represent non-invasive markers of CE and, in a near future, could be used to assess the endometrial quality in IVF., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: The aim of the present study was to assess the expression of miRNAs in the Vitreous Humor (VH) of patients with Macular Hole (MH) and Epiretinal Membrane (ERM) compared to a control group., Methods: In this prospective, comparative study, 2-ml of VH was extracted from the core of the vitreous chamber in consecutive patients who underwent standard vitrectomy for ERM and MH. RNA was extracted and TaqMan® Low Density Arrays (TLDAs) were used to profile the transcriptome of 754 miRNAs. Results were validated by single TaqMan® assays. Finally, we created a biological network of differentially expressed miRNA targets and their nearest neighbors., Results: Overall 10 eyes with MH, 16 eyes with idiopathic ERM and 6 controls were enrolled in the study. Profiling data identified 5 miRNAs differentially expressed in patients affected by MH and ERM with respect to controls. Four were downregulated (miR-19b, miR-24, miR-155, miR-451) and 1 was downregulated (miR-29a); TaqMan® assays of the VH of patients affected by MH and ERM, with respect to controls, showed that the most differentially expressed were miR-19b (FC -9.13, p:<0.00004), mir-24 (FC -7.52, p:<0.004) and miR-142-3p (FC -5.32, p:<0.011). Our network data showed that deregulation of differentially expressed miRNAs induces an alteration of several pathways associated with genes involved in both MH and ERM., Conclusion: The present study suggests that disregulation of miR-19b, miR-24 and miR-142-3p, might be related to the alterations that characterize patients affected by MH and ERM.

Neuroblastoma is the most common human extracranial solid tumor during infancy. Involvement of several miRNAs in its pathogenesis has been ascertained. Interestingly, most of their encoding genes reside in hypermethylated genomic regions: thus, their tumor suppressor function is normally disallowed in these tumors. To date, the therapeutic role of the demethylating agent 5'-Aza-2 deoxycytidine (5'-AZA) and its effects on miRNAome modulation in neuroblastoma have not been satisfactorily explored. Starting from a high-throughput expression profiling of 754 miRNAs and based on a proper selection, we focused on miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p as candidate miRNAs for our analysis. They resulted downregulated in four neuroblastoma cell lines with respect to normal adrenal gland. MiRNAs 29a-3p and 34b-3p also resulted downregulated in vivo in a murine neuroblastoma progression model. Unlike the amount of methylation of their encoding gene promoters, all these miRNAs were significantly overexpressed following treatment with 5'-AZA. Transfection with candidate miRNAs mimics significantly decreased neuroblastoma cells proliferation rate. A lower expression of miR-181c was significantly associated to a worse overall survival in a public dataset of 498 neuroblastoma samples (http://r2.amc.nl). Our data strongly suggest that CDK6, DNMT3A, DNMT3B are targets of miR-29a-3p, while CCNE2 and E2F3 are targets of miR-34b-3p. Based on all these data, we propose that miR-29a-3p, miR-34b-3p, miR-181c-5p and miR-517a-3p are disallowed tumor suppressor genes in neuroblastoma and suggest them as new therapeutic targets in neuroblastoma.

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., (© 2016 by the Society for the Study of Reproduction, Inc.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

NKT17 cells represent a functional subset of Vα14 invariant NKT (iNKT) cells with important effector functions in infections and autoimmune diseases. The mechanisms that drive NKT17 cell differentiation in the thymus are still largely unknown. The percentage of NKT17 cells has a high variability between murine strains due to differential thymic differentiation. For example, the NOD strain carries a high percentage and absolute numbers of NKT17 cells compared with other strains. In this study, we used the NOD mouse model to analyze what regulates NKT17 cell frequency in the thymus and peripheral lymphoid organs. In accordance with previous studies showing that the zinc finger transcription factor Th-POK is a key negative regulator of thymic NKT17 cell differentiation in the thymus, our data indicate that excessive NKT17 cell frequency in NOD mice correlates with defective Th-POK expression by thymic Vα14iNKT cells. Moreover, we found that Th-POK expression is under epigenetic regulation mediated by microRNA-133b whose expression is reduced in Vα14iNKT cells of NOD mice. We also demonstrated in a conditional knockout model of dendritic cell (DC) depletion (CD11cCreXDTA.B6 and CD11cCreRosa26DTA.NOD mice) that DCs play a crucial role in regulating Vα14iNKT cell maturation and their acquisition of an NKT17 cytokine secretion phenotype in the thymus. Overall, our data show that mechanisms regulating NKT17 cell differentiation are unique and completely different from those of Vα14iNKT cells. Specifically, we found that epigenetic regulation through microRNA-133b-regulated Th-POK expression and signals provided by DCs are fundamental for thymic NKT17 cell differentiation., (Copyright © 2016 by The American Association of Immunologists, Inc.)

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile.

Background: Tourette Syndrome (TS) is a highly prevalent childhood neuropsychiatric disorder (about 1 %), characterized by multiple motor and one or more vocal tics. The syndrome is commonly associated to comorbid conditions (e.g., Attention Deficit Hyperactivity Disorder and Obsessive Compulsive Disorder), which considerably aggravate clinical symptoms and complicate diagnosis and treatment. To date, TS molecular bases are unknown and its molecular diagnosis is unfeasible., Results: Due to their master role within cell networks and pathways both in physiology as in pathology, we sought to determine the transcriptome of circulating miRNAs in TS patients: by TaqMan Low Density Arrays, we profiled the expression in serum of 754 miRNAs in six TS patients and three unaffected controls (NCs) (discovery set). These data were validated by single TaqMan assays on serum from 52 TS patients and 15 NCs (validation set). Network and Gene-ontology analysis were performed by using Cytoscape and Babelomics server. We found that miR-429 is significantly underexpressed in TS patients with respect to NCs. Decreased serum levels of miR-429 allowed us to discriminate TS patients from NCs with 95 % of sensitivity and 42 % of specificity. Intriguingly, computational analysis of the network comprising miR-429 targets demonstrates their involvement in differentiation of midbrain and hindbrain and synaptic transmission., Conclusions: Our data open the way to further molecular characterization of TS and eventual identification of the corresponding genotypes. Circulating miR-429 may be immediately useful as sensitive molecular biomarker to support TS diagnosis, actually based only on DSM-V criteria.

Uveal melanoma (UM) represents approximately 5-6% of all melanoma diagnoses and up to 50% of patients succumb to their disease. Although several methods are available, accurate diagnosis is not always easily feasible because of potential accidents (e.g., intraocular hemorrhage). Based on the assumption that the profile of circulating miRNAs is often altered in human cancers, we verified whether UM patients showed different vitreous humor (VH) or serum miRNA profiles with respect to healthy controls. By using TaqMan Low Density Arrays, we analyzed 754 miRNAs from VH, vitreal exosomes, and serum of 6 UM patients and 6 healthy donors: our data demonstrated that the UM VH profile was unique and only partially overlapping with that from serum of the same patients. Whereas, 90% of miRNAs were shared between VH and vitreal exosomes, and their alterations in UM were statistically overlapped with those of VH and vitreal exosomes, suggesting that VH alterations could result from exosomal dysregulation. We report 32 miRNAs differentially expressed in UM patients in at least 2 different types of samples analyzed. We validated these data on an independent cohort of 12 UM patients. Most alterations were common to VH and vitreal exosomes (e.g., upregulation of miR-21,-34 a,-146a). Interestingly, miR-146a was upregulated in the serum of UM patients, as well as in serum exosomes. Upregulation of miR-21 and miR-146a was also detected in formalin-fixed, paraffin-embedded UM, suggesting that VH or serum alterations in UM could be the consequence of disregulation arising from tumoral cells. Our findings suggest the possibility to detect in VH and serum of UM patients "diagnostic" miRNAs released by the affected eye: based on this, miR-146a could be considered a potential circulating marker of UM.

Objective: To characterize well-represented microRNAs in human follicular fluid (FF) and to ascertain whether they are cargo of FF exosomes and whether they are involved in the regulation of follicle maturation., Design: FF exosomes were characterized by nanosight, flow cytometry, and exosome-specific surface markers. Expression microRNA profiles from total and exosomal FF were compared with those from plasma of the same women., Setting: University laboratory and an IVF center., Patient(s): Fifteen healthy women who had undergone intracytoplasmic sperm injection., Intervention(s): None., Main Outcome Measure(s): TaqMan low-density array to investigate the expression profile of 384 microRNAs; DataAssist and geNorm for endogenous control identification; significance analysis of microarrays to identify differentially expressed microRNAs; nanosight, flow-cytometry, and bioanalyzer for exosome characterization; bioinformatic tools for microRNAs target prediction, gene ontology, and pathway analysis., Result(s): We identified 37 microRNAs upregulated in FF as compared with plasma from the same women. Thirty-two were carried by microvesicles that showed the well-characterized exosomal markers CD63 and CD81. These FF microRNAs are involved in critically important pathways for follicle growth and oocyte maturation. Specifically, nine of them target and negatively regulate mRNAs expressed in the follicular microenvironment encoding inhibitors of follicle maturation and meiosis resumption., Conclusion(s): This study identified a series of exosomal microRNAs that are highly represented in human FF and are involved in follicular maturation. They could represent noninvasive biomarkers of oocyte quality in assisted reproductive technology., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The molecular bases of mammalian pancreatic α cells higher resistance than β 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 β (βTC1) 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 βTC1 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 IGF1Rβ, 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 IGF1Rβ and TNFα, is a major determinant of mammalian pancreatic α cells resistance to apoptosis induction by cytokines.

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.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has a master control role in various cancer-related biological processes as cell growth, proliferation, differentiation, migration, and apoptosis. It also regulates many transcription factors that control microRNAs (miRNAs) and their biosynthetic machinery. To investigate on the still poorly characterised global involvement of miRNAs within the pathway, we profiled the expression of 745 miRNAs in three colorectal cancer (CRC) cell lines after blocking the pathway with three different inhibitors. This allowed the identification of two classes of post-treatment differentially expressed (DE) miRNAs: (1) common DE miRNAs in all CRC lines after treatment with a specific inhibitor (class A); (2) DE miRNAs in a single CRC line after treatment with all three inhibitors (class B). By determining the molecular targets, biological roles, network position of chosen miRNAs from class A (miR-372, miR-663b, miR-1226*) and class B (miR-92a-1*, miR-135b*, miR-720), we experimentally demonstrated that they are involved in cell proliferation, migration, apoptosis, and globally affect the regulation circuits centred on MAPK/ERK signaling. Interestingly, the levels of miR-92a-1*, miR-135b*, miR-372, miR-720 are significantly higher in biopsies from CRC patients than in normal controls; they also are significantly higher in CRC patients with mutated KRAS than in those with wild-type genotypes (Wilcoxon test, p < 0.05): the latter could be a downstream effect of ERK pathway overactivation, triggered by KRAS mutations. Finally, our functional data strongly suggest the following miRNA/target pairs: miR-92a-1*/PTEN-SOCS5; miR-135b*/LATS2; miR-372/TXNIP; miR-663b/CCND2. Altogether, these results contribute to deepen current knowledge on still uncharacterized features of MAPK/ERK pathway, pinpointing new oncomiRs in CRC and allowing their translation into clinical practice and CRC therapy.

The relationship between therapeutic response and modifications of microRNA (miRNA) transcriptome in colorectal cancer (CRC) remains unknown. We investigated this issue by profiling the expression of 667 miRNAs in 2 human CRC cell lines, one sensitive and the other resistant to cetuximab (Caco-2 and HCT-116, respectively), through TaqMan real-time PCR. Caco-2 and HCT-116 expressed different sets of miRNAs after treatment. Specifically, 21 and 22 miRNAs were differentially expressed in Caco-2 or HCT-116, respectively (t test, P < 0.01). By testing the expression of differentially expressed miRNAs in CRC patients, we found that miR-146b-3p and miR-486-5p are more abundant in K-ras-mutated samples with respect to wild-type ones (Wilcoxon test, P < 0.05). Sixty-seven percent of differentially expressed miRNAs were involved in cancer, including CRC, whereas 19 miRNA targets had been previously reported to be involved in the cetuximab pathway and CRC. We identified 25 transcription factors putatively controlling these miRNAs, 11 of which have been already reported to be involved in CRC. On the basis of these data, we suggest that the downregulation of let-7b and let-7e (targeting K-ras) and the upregulation of miR-17* (a CRC marker) could be considered as candidate molecular markers of cetuximab resistance. Global network functional analysis (based on miRNA targets) showed a significant overrepresentation of cancer-related biological processes and networks centered on critical nodes involved in epidermal growth factor receptor internalization and ubiquitin-mediated degradation. The identification of miRNAs, whose expression is linked to the efficacy of therapy, should allow the ability to predict the response of patients to treatment and possibly lead to a better understanding of the molecular mechanisms of drug response., (© 2010 AACR.)

MicroRNAs (MIRs) perform critical regulatory functions within cell networks, both in physiology as well as in pathology. Through the positional gene candidate approach, we have identified three MIRs (MIR152, MIR200B, and MIR338) that are located in regions frequently altered in neuroblastoma (NB) and target mRNAs encoding proteins involved in cell proliferation, neuroblast differentiation, neuroblast migration, and apoptosis. Expression analysis in NB biopsies and NB cell lines showed that these MIRs are dysregulated. We have characterized a CpG island, close to the gene encoding MIR200B and hypermethylated in NB samples, that explains its negative regulation. Expression of MIR152, MIR200B, and MIR338 is specifically modulated in NB cell lines during differentiation and apoptosis. Functional genomic experiments through enforced expression of MIR200B and knockdown of MIR152 resulted in a significant decrease of the invasion activity of SH-SY5Y cells. Reconstruction of a NB network comprising MIR152, MIR200B, and MIR338 allowed us to confirm their role in the control of NB cell stemness and apoptosis: This suggests that altered regulation of these MIRs could have a role in NB pathogenesis by interfering with the molecular mechanisms, which physiologically control differentiation and death of neuroblasts. Accordingly, they could be considered as new NB biomarkers and potential targets of antagomirs or epigenetic therapies.