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.

An Uncharacterised lncRNA Coded by the ASAP1 Locus Is Downregulated in Serum of Type 2 Diabetes Mellitus Patients.

Diabetes mellitus (DM) is a complex and multifactorial disease characterised by high blood glucose. Type 2 Diabetes (T2D), the most frequent clinical condition accounting for about 90% of all DM cases worldwide, is a chronic disease with slow development usually affecting middle-aged or elderly individuals. T2D represents a significant problem of public health today because its incidence is constantly growing among both children and adults. It is also estimated that underdiagnosis prevalence would strongly further increase the real incidence of the disease, with about half of T2D patients being undiagnosed. Therefore, it is important to increase diagnosis accuracy. The current interest in RNA molecules (both protein- and non-protein-coding) as potential biomarkers for diagnosis, prognosis, and treatment lies in the ease and low cost of isolation and quantification with basic molecular biology techniques. In the present study, we analysed the transcriptome in serum samples collected from T2D patients and unaffected individuals to identify potential RNA-based biomarkers. Microarray-based profiling and subsequent validation using Real-Time PCR identified an uncharacterised long non-coding RNA (lncRNA) transcribed from the ASAP1 locus as a potential diagnostic biomarker. ROC curve analysis showed that a molecular signature including the lncRNA and the clinicopathological parameters of T2D patients as well as unaffected individuals showed a better diagnostic performance compared with the glycated haemoglobin test (HbA1c). This result suggests that the application of this biomarker in clinical practice would help to improve the diagnosis, and therefore the clinical management, of T2D patients. The proposed biomarker would be useful in the context of predictive, preventive, and personalised medicine (3PM/PPPM).

FUS driven circCNOT6L biogenesis in mouse and human spermatozoa supports zygote development.

Circular RNA (circRNA) biogenesis requires a backsplicing reaction, promoted by inverted repeats in cis-flanking sequences and trans factors, such as RNA-binding proteins (RBPs). Among these, FUS plays a key role. During spermatogenesis and sperm maturation along the epididymis such a molecular mechanism has been poorly explored. With this in mind, we chose circCNOT6L as a study case and wild-type (WT) as well as cannabinoid receptor type-1 knock-out (Cb1-/-) male mice as animal models to analyze backsplicing mechanisms. Our results suggest that spermatozoa (SPZ) have an endogenous skill to circularize mRNAs, choosing FUS as modulator of backsplicing and under CB1 stimulation. A physical interaction between FUS and CNOT6L as well as a cooperation among FUS, RNA Polymerase II (RNApol2) and Quaking (QKI) take place in SPZ. Finally, to gain insight into FUS involvement in circCNOT6L biogenesis, FUS expression was reduced through RNA interference approach. Paternal transmission of FUS and CNOT6L to oocytes during fertilization was then assessed by using murine unfertilized oocytes (NF), one-cell zygotes (F) and murine oocytes undergoing parthenogenetic activation (PA) to exclude a maternal contribution. The role of circCNOT6L as an active regulator of zygote transition toward the 2-cell-like state was suggested using the Embryonic Stem Cell (ESC) system. Intriguingly, human SPZ exactly mirror murine SPZ.

Anatomical distribution of cancer stem cells between enhancing nodule and FLAIR hyperintensity in supratentorial glioblastoma: time to recalibrate the surgical target?

It is ge nerally accepted that glioblastoma (GBM) arise from cancer stem cells (CSC); however, there is little evidence on their anatomical distribution. We investigated the expression and distribution of SOX-2-positive and CD133-positive CSCs both in the enhancing nodule (EN) of GBM and in the FLAIR hyperintensity zones on a surgical, histopathological series of 33 GBMs. The inclusion criterion was the intraoperative sampling of different tumor regions individualized, thanks to neuronavigation and positivity to intraoperative fluorescence with the use of 5-aminolevulinic acid (5-ALA). Thirty-three patients (20 males and 13 females with a mean age at diagnosis of 56 years) met the inclusion criterion. A total of 109 histological samples were evaluated, 52 for ENs and 57 for FLAIR hyperintensity zone. Considering the quantitative distribution of levels of intensity of staining (IS), ES (extent score), and immunoreactivity score (IRS), no difference was found between ENs and FLAIR regions for both the SOX-2 biomarker (respectively, IS p = 0.851, ES p = 0.561, IRS p = 1.000) and the CD133 biomarker (IS p = 0.653, ES p = 0.409, IRS p = 0.881). This evidence suggests to recalibrate the target of surgery for FLAIRECTOMY and 5-ALA could improve the possibility to achieve this goal.

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.

The Immunohistochemical Expression of the Serine and Arginine-Rich Splicing Factor 1 (SRSF1) Is a Predictive Factor of the Recurrence of Basal Cell Carcinoma: A Preliminary Study on a Series of 52 Cases.

Background and Objectives: Basal cell carcinomas (BCCs) are the most frequent skin tumors; although they usually exhibit a good prognosis, it has been reported that there is a 2-8% rate of local recurrence of surgically-excised BCCs, even in the presence of tumor-free surgical margins. Several histological and clinical risk factors have been associated with a higher risk of local relapse; however, the exact pathogenetic mechanisms that regulate the local recurrence of these tumors are still to be elucidated. The serine and arginine-rich splicing factor 1 (SRSF1) is an RNA-binding protein whose oncogenic function has been described in numerous forms of human cancers, including brain, lung, and prostate tumors. We evaluated the correlation between SRSF1 immunoexpression and the local recurrence of BCCs. Materials and Methods: Fifty-two cases of surgically excised BCCs with free-tumor margins (10 high-risk and 42 low-risk variants), for which follow-up data were available, were selected. Local recurrence occurred in only 5 cases. Results: We found high and low immunoexpressions of SRSF1 in 18 and 34 cases, respectively. A statistically significant association between high SRSF1 immunoexpression and the local recurrence of BCC was found (p = 0.0433). Conclusions: Our immunohistochemical results suggest an active role of SRSF1 in inducing a local recurrence of BCCs; however, further studies on a larger series are needed to validate our findings.

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.

Dysregulation of miR-15a-5p, miR-497a-5p and miR-511-5p Is Associated with Modulation of BDNF and FKBP5 in Brain Areas of PTSD-Related Susceptible and Resilient Mice.

Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder occurring in susceptible individuals following a traumatic event. Understanding the mechanisms subserving trauma susceptibility/resilience is essential to develop new effective treatments. Increasing evidence suggests that non-coding RNAs, such as microRNAs (miRNAs), may play a prominent role in mediating trauma susceptibility/resilience. In this study, we evaluated the transcriptional expression of two key PTSD-related genes (FKBP5 and BDNF) and the relative targeting miRNAs (miR-15a-5p, miR-497a-5p, miR-511-5p, let-7d-5p) in brain areas of PTSD-related susceptible and resilient mice identified through our recently developed mouse model of PTSD (arousal-based individual screening (AIS) model). We observed lower transcript levels of miR-15a-5p, miR-497a-5p, and miR-511a-5p in the hippocampus and hypothalamus of susceptible mice compared to resilient mice, suggesting that the expression of these miRNAs could discriminate the two different phenotypes of stress-exposed mice. These miRNA variations could contribute, individually or synergically, to the inversely correlated transcript levels of FKBP5 and BDNF. Conversely, in the medial prefrontal cortex, downregulation of miR-15a-5p, miR-511-5p, and let-7d-5p was observed both in susceptible and resilient mice, and not accompanied by changes in their mRNA targets. Furthermore, miRNA expression in the different brain areas correlated to stress-induced behavioral scores (arousal score, avoidance-like score, social memory score and PTSD-like score), suggesting a linear connection between miRNA-based epigenetic modulation and stress-induced phenotypes. Pathway analysis of a miRNA network showed a statistically significant enrichment of molecular processes related to PTSD and stress. In conclusion, our results indicate that PTSD susceptibility/resilience might be shaped by brain-area-dependent modulation of miRNAs targeting FKBP5, BDNF, and other stress-related genes.

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.

Specific Signatures of Serum miRNAs as Potential Biomarkers to Discriminate Clinically Similar Neurodegenerative and Vascular-Related Diseases.

Neurodegenerative diseases (NDs) are age-dependent; among them, Alzheimer's disease (AD) and Parkinson's disease (PD) are the most frequent. Similarly, cerebrovascular damage can induce the development of vascular-related disorders that share common features with AD and PD, respectively, named vascular dementia (VD) and vascular parkinsonism (VP). To date, ND diagnosis is mainly clinical; therefore, since these disorders show similar symptoms, their correct discrimination may be difficult. We detected 23 ND-associated microRNAs (miRNAs) by literature mining and investigated their serum expression in a cohort of 139 patients including AD, PD, VD, and VP patients and healthy controls. TaqMan RT-PCR data showed that miR-23a upregulation was associated with an ongoing neurodegenerative process, similar to miR-22* and miR-29a, while let-7d, miR-15b, miR-24, miR-142-3p, miR-181c, and miR-222 showed an altered expression in Parkinson-like phenotypes, as well as miR-34b, miR-125b, and miR-130b in Alzheimer-like disorders. By computing logistic regression models and ROC curves, we identified signatures of neuro-miRNAs specific for each disease, showing good diagnostic performance. Interestingly, we found that miR-23a, miR-29a, miR-34b, and miR-125b exhibited a different distribution between exosomes and vesicle-free serum, suggesting a heterogeneity of secretion for these miRNAs. Our results suggest that miRNA signatures could discriminate in a non-invasive manner neurodegenerative disorders, thus improving clinical diagnoses.

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.

Serum coding and non-coding RNAs as biomarkers of NAFLD and fibrosis severity.

BACKGROUND & AIMS: In patients with non-alcoholic fatty liver disease (NAFLD), liver biopsy is the gold standard to detect non-alcoholic steatohepatitis (NASH) and stage liver fibrosis. We aimed to identify differentially expressed mRNAs and non-coding RNAs in serum samples of biopsy-diagnosed mild and severe NAFLD patients with respect to controls and to each other. METHODS: We first performed a whole transcriptome analysis through microarray (n = 12: four Control: CTRL; four mild NAFLD: NAS ≤ 4 F0; four severe NAFLD NAS ≥ 5 F3), followed by validation of selected transcripts through real-time PCRs in an independent internal cohort of 88 subjects (63 NAFLD, 25 CTRL) and in an external cohort of 50 NAFLD patients. A similar analysis was also performed on liver biopsies and HepG2 cells exposed to oleate:palmitate or only palmitate (cellular model of NAFL/NASH) at intracellular/extracellular levels. Transcript correlation with histological/clinical data was also analysed. RESULTS: We identified several differentially expressed coding/non-coding RNAs in each group of the study cohort. We validated the up-regulation of UBE2V1, BNIP3L mRNAs, RP11-128N14.5 lncRNA, TGFB2/TGFB2-OT1 coding/lncRNA in patients with NAS ≥ 5 (vs NAS ≤ 4) and the up-regulation of HBA2 mRNA, TGFB2/TGFB2-OT1 coding/lncRNA in patients with Fibrosis stages = 3-4 (vs F = 0-2). In in vitro models: UBE2V1, RP11-128N14.5 and TGFB2/TGFB2-OT1 had an increasing expression trend ranging from CTRL to oleate:palmitate or only palmitate-treated cells both at intracellular and extracellular level, while BNIP3L was up-regulated only at extracellular level. UBE2V1, RP11-128N14.5, TGFB2/TGFB2-OT1 and HBA2 up-regulation was also observed at histological level. UBE2V1, RP11-128N14.5, BNIP3L and TGFB2/TGFB2-OT1 correlated with histological/biochemical data. Combinations of TGFB2/TGFB2-OT1 + Fibrosis Index based on the four factors (FIB-4) showed an Area Under the Curve (AUC) of 0.891 (P = 3.00E-06) or TGFB2/TGFB2-OT1 + Fibroscan (AUC = 0.892, P = 2.00E-06) improved the detection of F = 3-4 with respect to F = 0-2 fibrosis stages. CONCLUSIONS: We identified specific serum coding/non-coding RNA profiles in severe and mild NAFLD patients that possibly mirror the molecular mechanisms underlying NAFLD progression towards NASH/fibrosis. TGFB2/TGFB2-OT1 detection improves FIB-4/Fibroscan diagnostic performance for advanced fibrosis discrimination.

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.

Non-Coding RNAs in Endometrial Physiopathology.

The Human Genome Project led to the discovery that about 80% of our DNA is transcribed in RNA molecules. Only 2% of the human genome is translated into proteins, the rest mostly produces molecules called non-coding RNAs, which are a heterogeneous class of RNAs involved in different steps of gene regulation. They have been classified, according to their length, into small non-coding RNAs and long non-coding RNAs, or to their function, into housekeeping non-coding RNAs and regulatory non-coding RNAs. Their involvement has been widely demonstrated in all cellular processes, as well as their dysregulation in human pathologies. In this review, we discuss the function of non-coding RNAs in endometrial physiology, analysing their involvement in embryo implantation. Moreover, we explore their role in endometrial pathologies such as endometrial cancer, endometriosis and chronic endometritis.

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.