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.

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).

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.

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.

Resveratrol Treatment Induces Mito-miRNome Modification in Follicular Fluid from Aged Women with a Poor Prognosis for In Vitro Fertilization Cycles.

Advanced maternal age impairs reproductive performance, influencing the quantity and the quality of oocytes. Mitochondria dysfunction seems to play a decisive role in conditioning the quality of the female gamete. Different in vitro and in vivo studies, demonstrated the antioxidant and anti-inflammatory activities of Resveratrol and its ability to improve mitochondria function even if the exact mechanism of action has not yet been demonstrated in human oocytes. In this paper, by retrospective analysis, we evaluated follicular fluid (FF) miRNome modification in aged women with a poor ovarian reserve receiving a resveratrol-based supplement the three months before the in vitro Fertilization (IVF) cycle. We found 13 differentially expressed microRNAs (miRNAs) in women treated with resveratrol and specifically miR-125b-5p, miR-132-3p, miR-19a-3p, miR-30a-5p and miR-660-5p, regulating mitochondrial proteins, are able to control metabolism and mitochondrial biogenesis. MiRNA expression differences, observed after resveratrol treatment in FF from women with a poor prognosis for IVF, demonstrated that resveratrol may act on mitomiRNAs to improve follicular microenvironment by transcriptomic and proteomic modifications in granulosa cells.

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.

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.

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.

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.

Do Extracellular RNAs Provide Insight into Uveal Melanoma Biology?

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults, showing a high mortality due to metastasis. Although it is considered a rare disease, a growing number of papers have reported altered levels of RNAs (i.e., coding and non-coding RNAs) in cancerous tissues and biological fluids from UM patients. The presence of circulating RNAs, whose dysregulation is associated with UM, paved the way to the possibility of exploiting it for diagnostic and prognostic purposes. However, the biological meaning and the origin of such RNAs in blood and ocular fluids of UM patients remain unexplored. In this review, we report the state of the art of circulating RNAs in UM and debate whether the amount and types of RNAs measured in bodily fluids mirror the RNA alterations from source cancer cells. Based on literature data, extracellular RNAs in UM patients do not represent, with rare exceptions, a snapshot of RNA dysregulations occurring in cancerous tissues, but rather the complex and heterogeneous outcome of a systemic dysfunction, including immune system activity, that modifies the mechanisms of RNA delivery from several cell types.

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.

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.

A Novel Silicon Platform for Selective Isolation, Quantification, and Molecular Analysis of Small Extracellular Vesicles.

INTRODUCTION: Small extracellular vesicles (sEVs), thanks to their cargo, are involved in cellular communication and play important roles in cell proliferation, growth, differentiation, apoptosis, stemness and embryo development. Their contribution to human pathology has been widely demonstrated and they are emerging as strategic biomarkers of cancer, neurodegenerative and cardiovascular diseases, and as potential targets for therapeutic intervention. However, the use of sEVs for medical applications is still limited due to the selectivity and sensitivity limits of the commonly applied approaches. METHODS: Novel sensing solutions based on nanomaterials are arising as strategic tools able to surpass traditional sensor limits. Among these, Si nanowires (Si NWs), realized with cost-effective industrially compatible metal-assisted chemical etching, are perfect candidates for sEV detection. RESULTS: In this paper, the realization of a selective sensor able to isolate, concentrate and quantify specific vesicle populations, from minimal volumes of biofluid, is presented. In particular, this Si NW platform has a detection limit of about 2×105 sEVs/mL and was tested with follicular fluid and blastocoel samples. Moreover, the possibility to detach the selectively isolated sEVs allowing further analyses with other approaches was demonstrated by SEM analysis and several PCRs performed on the RNA content of the detached sEVs. DISCUSSION: This platform overcomes the limit of detection of traditional methods and, most importantly, preserves the biological content of sEVs, opening the route toward a reliable liquid biopsy analysis.

VECTOR: An Integrated Correlation Network Database for the Identification of CeRNA Axes in Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults and, although its genetic background has been extensively studied, little is known about the contribution of non-coding RNAs (ncRNAs) to its pathogenesis. Indeed, its competitive endogenous RNA (ceRNA) regulatory network comprising microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and mRNAs has been insufficiently explored. Thanks to UM findings from The Cancer Genome Atlas (TCGA), it is now possible to statistically elaborate these data to identify the expression relationships among RNAs and correlative interaction data. In the present work, we propose the VECTOR (uVeal mElanoma Correlation NeTwORk) database, an interactive tool that identifies and visualizes the relationships among RNA molecules, based on the ceRNA model. The VECTOR database contains: i) the TCGA-derived expression correlation values of miRNA-mRNA, miRNA-lncRNA and lncRNA-mRNA pairs combined with predicted or validated RNA-RNA interactions; ii) data of sense-antisense sequence overlapping; iii) correlation values of Transcription Factor (TF)-miRNA, TF-lncRNA, and TF-mRNA pairs associated with ChiPseq data; iv) expression data of miRNAs, lncRNAs and mRNAs both in UM and physiological tissues. The VECTOR web interface can be queried, by inputting the gene name, to retrieve all the information about RNA signaling and visualize this as a graph. Finally, VECTOR provides a very detailed picture of ceRNA networks in UM and could be a very useful tool for researchers studying RNA signaling in UM. The web version of Vector is freely available at the URL reported at the end of the Introduction.

Serum Extracellular Vesicle-Derived circHIPK3 and circSMARCA5 Are Two Novel Diagnostic Biomarkers for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most frequent and deadly human brain cancer. Early diagnosis through non-invasive biomarkers may render GBM more easily treatable, improving the prognosis of this currently incurable disease. We suggest the use of serum extracellular vesicle (sEV)-derived circular RNAs (circRNAs) as highly stable minimally invasive diagnostic biomarkers for GBM diagnosis. EVs were isolated by size exclusion chromatography from sera of 23 GBM and 5 grade 3 glioma (GIII) patients, and 10 unaffected controls (UC). The expression of two candidate circRNAs (circSMARCA5 and circHIPK3) was assayed by droplet digital PCR. CircSMARCA5 and circHIPK3 were significantly less abundant in sEVs from GBM patients with respect to UC (fold-change (FC) of -2.15 and -1.92, respectively) and GIII (FC of -1.75 and -1.4, respectively). Receiver operating characteristic curve (ROC) analysis, based on the expression of sEV-derived circSMARCA5 and circHIPK3, allowed us to distinguish GBM from UC (area under the curve (AUC) 0.823 (0.667-0.979) and 0.855 (0.704 to 1.000), with a 95% confidence interval (CI), respectively). Multivariable ROC analysis, performed by combining the expression of sEV-derived circSMARCA5 and circHIPK3 with preoperative neutrophil to lymphocyte (NLR), platelet to lymphocyte (PLR) and lymphocyte to monocyte (LMR) ratios, three known diagnostic and prognostic GBM markers, allowed an improvement in the GBM diagnostic accuracy (AUC 0.901 (0.7912 to 1.000), 95% CI). Our data suggest sEV-derived circSMARCA5 and circHIPK3 as good diagnostic biomarkers for GBM, especially when associated with preoperative NLR, PLR and LMR.

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.

Peritumoral Microenvironment in High-Grade Gliomas: From FLAIRectomy to Microglia-Glioma Cross-Talk.

Cellular composition and molecular signatures of the glioma core compared with infiltrative margins are different, and it is well known that the tumor edge is enriched in microglia. In this review of the literature, we summarize the role of the peritumoral area in high-grade gliomas (HGGs) from surgical and biological points of view. There is evidence on the dual role of microglia in HGGs-a scavenger-tumoricidal role when microglia are activated in an M1 phenotype and a role favoring tumor growth and infiltration/migration when microglia are activated in an M2 phenotype. Microglia polarization is mediated by complex pathways involving cross-talk with glioma cells. In this scenario, extracellular vesicles and their miRNA cargo seem to play a central role. The switch to a specific phenotype correlates with prognosis and the pathological assessment of a specific microglial setting can predict a patient's outcome. Some authors have designed an engineered microglial cell as a biologically active vehicle for the delivery of intraoperative near-infrared fluorescent dye with the aim of helping surgeons detect peritumoral infiltrated areas during resection. Furthermore, the pharmacological modulation of microglia-glioma cross-talk paves the way to more effective therapies.

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.

LncRNA LINC00518 Acts as an Oncogene in Uveal Melanoma by Regulating an RNA-Based Network.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults; little is known about the contribution of non-coding RNAs (ncRNAs) to UM pathogenesis. Competitive endogenous RNA (ceRNA) networks based on RNA-RNA interactions regulate physiological and pathological processes. Through a combined approach of in silico and experimental biology, we investigated the expression of a set of long non-coding RNAs (lncRNAs) in patient biopsies, identifying LINC00518 as a potential oncogene in UM. The detection of LINC00518 dysregulation associated with several in vitro functional assays allowed us to investigate its ceRNA regulatory network and shed light on its potential involvement in cancer-related processes, such as epithelial to mesenchymal transition (EMT) and CoCl2-induced hypoxia-like response. In vitro transient silencing of LINC00518 impaired cell proliferation and migration, and affected mRNA expression of LINGO2, NFIA, OTUD7B, SEC22C, and VAMP3. A "miRNA sponge" and "miRNA protector" model have been hypothesized for LINC00518-induced regulation of mRNAs. In vitro inhibition of MITF suggested its role as a potential activator of LINC00518 expression. Comprehensively, LINC00518 may be considered a new oncogene in UM and a potential target for RNA-based therapeutic approaches.

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.