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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. [ABSTRACT FROM AUTHOR]

Simple Summary: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive primary liver cancer leading to death in 10–15% of cases. In recent years, the iCCA worldwide incidence has been increasing, and research is currently focused on identifying valuable diagnostic biomarkers and developing specific therapies to counteract the disease despite its high heterogeneity. The use of cutting-edge and broad-spectrum applications like omics represents a successful approach to studying the intricate pathobiology of iCCA. In this review, we discuss how both single- and multi-omics studies can pave the way for identifying potential biomarkers, and more importantly, for stratifying iCCA patients. This stratification aims to enhance the therapeutic intervention through personalized medicine (PM). Intrahepatic cholangiocarcinoma (iCCA) is recognized worldwide as the second leading cause of morbidity and mortality among primary liver cancers, showing a continuously increasing incidence rate in recent years. iCCA aggressiveness is revealed through its rapid and silent intrahepatic expansion and spread through the lymphatic system leading to late diagnosis and poor prognoses. Multi-omics studies have aggregated information derived from single-omics data, providing a more comprehensive understanding of the phenomena being studied. These approaches are gradually becoming powerful tools for investigating the intricate pathobiology of iCCA, facilitating the correlation between molecular signature and phenotypic manifestation. Consequently, preliminary stratifications of iCCA patients have been proposed according to their "omics" features opening the possibility of identifying potential biomarkers for early diagnosis and developing new therapies based on personalized medicine (PM). The focus of this review is to provide new and advanced insight into the molecular pathobiology of the iCCA, starting from single- to the latest multi-omics approaches, paving the way for translating new basic research into therapeutic practices. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) represents an increasing health burden. Evidence suggests the importance of miRNA in diagnosing CKD, yet the reports are inconsistent. This study aimed to determine novel miRNA biomarkers and potential therapeutic targets from hypothesis-free miRNA profiling studies in human and murine CKDs. Comprehensive literature searches were conducted on five databases. Subgroup analyses of kidney diseases, sample types, disease stages, and species were conducted. A total of 38 human and 12 murine eligible studies were analyzed using Robust Rank Aggregation (RRA) and vote-counting analyses. Gene set enrichment analyses of miRNA signatures in each kidney disease were conducted using DIANA-miRPath v4.0 and MIENTURNET. As a result, top target genes, Gene Ontology terms, the interaction network between miRNA and target genes, and molecular pathways in each kidney disease were identified. According to vote-counting analysis, 145 miRNAs were dysregulated in human kidney diseases, and 32 were dysregulated in murine CKD models. By RRA, miR-26a-5p was significantly reduced in the kidney tissue of Lupus nephritis (LN), while miR-107 was decreased in LN patients' blood samples. In both species, epithelial-mesenchymal transition, Notch, mTOR signaling, apoptosis, G2/M checkpoint, and hypoxia were the most enriched pathways. These miRNA signatures and their target genes must be validated in large patient cohort studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Simple Summary: Uveal melanoma (UM) is a rare eye cancer with a high mortality rate due to metastases, leading to death in up to 50% of patients within 10 years from UM diagnosis. Moreover, patients show a median survival of 6 to 12 months after metastasis diagnosis. UM and cutaneous melanoma (CM) have the same melanocytic origin; however, they are very different in terms of molecular alterations and biological behavior. In this review, we will discuss the complex genetic and non-coding RNA-based epigenetic landscapes underlying the transformation, progression, and dissemination of UM. This knowledge will pave the way for the future identification of new biomarkers of the pathology and therapeutic targets. Uveal melanoma (UM) is the most common intraocular malignant tumor and the most frequent melanoma not affecting the skin. While the rate of UM occurrence is relatively low, about 50% of patients develop metastasis, primarily to the liver, with lethal outcome despite medical treatment. Notwithstanding that UM etiopathogenesis is still under investigation, a set of known mutations and chromosomal aberrations are associated with its pathogenesis and have a relevant prognostic value. The most frequently mutated genes are BAP1, EIF1AX, GNA11, GNAQ, and SF3B1, with mutually exclusive mutations occurring in GNAQ and GNA11, and almost mutually exclusive ones in BAP1 and SF3B1, and BAP1 and EIF1AX. Among chromosomal aberrations, monosomy of chromosome 3 is the most frequent, followed by gain of chromosome 8q, and full or partial loss of chromosomes 1 and 6. In addition, epigenetic mechanisms regulated by non-coding RNAs (ncRNA), namely microRNAs and long non-coding RNAs, have also been investigated. Several papers investigating the role of ncRNAs in UM have reported that their dysregulated expression affects cancer-related processes in both in vitro and in vivo models. This review will summarize current findings about genetic mutations, chromosomal aberrations, and ncRNA dysregulation establishing UM biology. [ABSTRACT FROM AUTHOR]

Lanthanide-doped yttrium oxide nanoparticles can display selective upconversion properties, rendering them invaluable in the field of nanomedicine for both sensing and diagnostics. Different syntheses of Er:Y2O3 and Nd:Y2O3 nanoparticles (NPs) were studied and optimized to obtain small particles of regular shape and good crystallinity. The morphological and compositional characterizations of the nanoparticles were obtained with different techniques and showed that both Er:Y2O3 and Nd:Y2O3 NPs were well dispersed, with dimensions of the order of a few tens of nanometers. The photoluminescence and cathodoluminescence measurements showed that both Er:Y2O3 and Nd:Y2O3 NPs had good emission as well as upconversion. The nanophosphors were functionalized by a pegylation procedure to suppress unwanted reactions of the NPs with other biological components, making the NP systems biocompatible and the NPs soluble in water and well dispersed. The pegylated core/shell nanoparticles showed the same morphological and optical characteristics as the core, promoting their strategic role as photoactive material for theragnostics and biosensing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are the most heterogeneous class of non-protein-coding RNAs involved in a broad spectrum of molecular mechanisms controlling genome function, including the generation of complex networks of RNA-RNA competitive interactions. Accordingly, their dysregulation contributes to the onset of many tumors, including colorectal cancer (CRC). Through a combination of in silico approaches (statistical screening of expression datasets) and in vitro analyses (enforced expression, artificial inhibition, or activation of pathways), we identified LINC00483 as a potential tumor suppressor lncRNA in CRC. LINC00483 was downregulated in CRC biopsies and metastases and its decreased levels were associated with severe clinical features. Inhibition of the MAPK pathway and cell cycle arrest by starvation induced an upregulation of LINC00483, while the epithelial to mesenchymal transition activation by TGFβ-1 and IL-6 caused its down-modulation. Moreover, enforced expression of LINC00483 provoked a slowing down of cell migration rate without affecting cell proliferation. Since LINC00483 was predominantly cytoplasmic, we hypothesized a "miRNA sponge" role for it. Accordingly, we computationally reconstructed the LINC00483/miRNA/mRNA axes and evaluated the expression of mRNAs in different experimental conditions inducing LINC00483 alteration. By this approach, we identified a set of mRNAs sharing the miRNA response elements with LINC00483 and modulated in accordance with it. Moreover, we found that LINC00483 is potentially under negative control of transcription factor HNF4α. In conclusion, we propose that LINC00483 is a tumor suppressor in CRC that, through an RNA-RNA network, may control cell migration and participate in proliferation signaling. [ABSTRACT FROM AUTHOR]

Choroidal melanoma (CM), despite its rarity, is the most frequent intraocular malignancy. Over time, several histological variants of CM have been distinguished, including spindle A and B cell, fascicular, epithelioid and necrotic type. However, they have been progressively abandoned as having no prognostic value and currently, the American Joint Committee of Cancer (AJCC) classification identifies three CM cell types: spindle, epithelioid and mixed cell type. Other rare histological variants of CM include: (i) diffuse melanoma; (ii) clear cell; and (iii) balloon cell melanoma. Immunohistochemically, CMs are stained with Human Melanoma Black 45 (HMB45) antigen, S-100 protein, Melan-A (also known as melanoma antigen recognized by T cells 1/MART-1), melanocyte inducing transcription factor (MITF), tyrosinase, vimentin, and Sex determining region Y-Box 10 (SOX10). Several genetic and histopathological prognostic factors of CM have been reported in the literature, including epithelioid cell type, TNM staging, extraocular extension, monosomy 3 and 6p gain and loss of BAP-1 gene. The aim of this review was to summarize the histopathological, immunohistochemical and genetic features of CM, establishing "the state of the art" and providing colleagues with practical tools to promptly deal with patients affected by this rare malignant neoplasm. [ABSTRACT FROM AUTHOR]

Transthyretin variant amyloidosis (ATTRv) is a rare autosomal dominant disease characterized by the accumulation of amyloid in many organs, mostly causing a sensory-motor neuropathy, cardiomyopathy, and dysautonomia. The aim of the study was to report microRNAs (miRNAs) expression profile identified in the blood of ATTRv patients. Ten ATTRv patients, 10 asymptomatic carriers of transthyretin variant (TTRv), 10 patients with Charcot-Marie-Tooth (CMT) disease, and 10 healthy controls were studied. Human Schwann cells cultures were used to study the regulatory effects of miR-150-5p on the expression of cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). ATTRv patients had 33 miRNAs up-regulated and 48 down-regulated versus healthy controls; 9 miRNAs were up-regulated and 30 down-regulated versus CMT patients; 19 miRNAs were up-regulated and 38 down-regulated versus asymptomatic TTRv carriers. Twelve out of the 19 upregulated miRNAs had a fold increase higher than 100. The validation experiment indicated miR-150-5p as a valuable biomarker to differentiate ATTRv patients from asymptomatic TTRv carriers (AUC: 0.9728; p < 0.0001). Schwann cells culture model demonstrated that miR-150-5p is a powerful negative regulator of CREB, BDNF, and NGF genes. Identification of deregulated miRNAs can help in understanding the complex pathomechamism underlying the development of ATTRv and related multisystemic pathology. Further investigations are needed on the role of circulating miR-150-5p to predict the shift of TTRv carriers from an asymptomatic status to symptoms appearance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer's disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-β (Aβ). Methods: We used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aβ, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs). Results: In the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aβ treatment. On the contrary, in endothelial monocultures, Aβ induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aβ. This modulation by Aβ was not observed in endothelial monocultures. In addition, endothelial expression of β-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aβ only in the co-culture model. miR-200b, increased in astrocytes following Aβ treatment and may represent one of the factors involved in the control of Gnt-III expression. Conclusion: These data point out that, at least in the early phases of Aβ exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer. [ABSTRACT FROM AUTHOR]

Concussion is difficult to diagnose, particularly when symptoms are atypical or late in presenting. An accurate and timely initial assessment is crucial for clinical management. Cerebral spinal fluid (CSF) and blood markers of traumatic brain injury show promising results but their clinical applicability in concussion has significant limitations. In the study, we explored saliva as a new source of biomarkers of concussion. Saliva samples of concussed players were collected after 48–72 h from concussion and analyzed by high-throughput technologies. A discovery group of 10 concussed rugby professional and semiprofessional athletes and 10 non-concussed matched controls was used for the analysis of 92 inflammatory proteins by the Proseek-Multiplex-Inflammation technology. In addition, saliva samples of 6 concussed and 6 non-concussed athletes were used to screen 800 human microRNAs (miRNAs) by the Nanostring Technology. The results were then validated by RT-qPCR in an enlarged cohort (validation group) comprising 22 concussed athletes. Results showed, no significant variations of the 65 inflammatory proteins detected in saliva between groups but 5 microRNAs, miR-27b-3p (p = 0.016), let-7i-5p (p = 0.001), miR-142-3p (p = 0.008), miR-107 (p = 0.028), miR-135b-5p (p = 0.017) significantly upregulated in concussed athletes. Univariate ROC curve analysis showed that the differentially expressed miRNAs could be considered good classifiers of concussion. Further analyses showed significant correlation between these microRNAs and Reaction Time component of the ImPACT concussion assessment tool. In addition, biocomputation analysis predicted the involvement of these microRNAs in important biological processes that might be related to trauma, such as response to hypoxia, cell death, neurogenesis, axon repair and myelination. Ease of access and non-invasiveness of saliva samples make these biomarkers particularly suitable for concussion assessment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Over the past few years, noncoding RNAs (ncRNAs) have been extensively studied because of the significant biological roles that they play in regulation of cellular mechanisms. ncRNAs are associated to higher eukaryotes complexity; accordingly, their dysfunction results in pathological phenotypes, including cancer. To date, most research efforts have been mainly focused on how ncRNAs could modulate the expression of protein-coding genes in pathological phenotypes. However, recent evidence has shown the existence of an unexpected interplay among ncRNAs that strongly influences cancer development and progression. ncRNAs can interact with and regulate each other through various molecular mechanisms generating a complex network including different species of RNAs (e.g., mRNAs, miRNAs, lncRNAs, and circRNAs). Such a hidden network of RNA-RNA competitive interactions pervades and modulates the physiological functioning of canonical protein-coding pathways involved in proliferation, differentiation, and metastasis in cancer. Moreover, the pivotal role of ncRNAs as keystones of network structural integrity makes them very attractive and promising targets for innovative RNA-based therapeutics. In this review we will discuss: (1) the current knowledge on complex crosstalk among ncRNAs, with a special focus on cancer; and (2) the main issues and criticisms concerning ncRNAs targeting in therapeutics. [ABSTRACT FROM AUTHOR]

Given its prevalence and social impact, Autism Spectrum Disorder (ASD) is drawing much interest. Molecular basis of ASD is heterogeneous and only partially known. Many factors, including disorders comorbid with ASD, like TS (Tourette Syndrome), complicate ASD behavior-based diagnosis and make it vulnerable to bias. To further investigate ASD etiology and to identify potential biomarkers to support its precise diagnosis, we used TaqMan Low Density Array technology to profile serum miRNAs from ASD, TS, and TS+ASD patients, and unaffected controls (NCs). Through validation assays in 30 ASD, 24 TS, and 25 TS+ASD patients and 25 NCs, we demonstrated that miR-140-3p is upregulated in ASD vs.: NC, TS, and TS+ASD (Tukey's test, p-values = 0.03, = 0.01, < 0.0001, respectively). 1Ct values for miR-140-3p and YGTSS (Yale Global Tic Severity Scale) scores are positively correlated (Spearman r = 0.33; Benjamini-Hochberg p = 0.008) and show a linear relationship (p = 0.002). Network functional analysis showed that nodes controlled by miR-140-3p, especially CD38 and NRIP1 which are its validated targets, are involved in processes convergingly dysregulated in ASD, such as synaptic plasticity, immune response, and chromatin binding. Biomarker analysis proved that serum miR-140-3p can discriminate among: (1) ASD and NC (Area under the ROC curve, AUC: 0.70; sensitivity: 63.33%; specificity: 68%); (2) ASD and TS (AUC: 0.72; sensitivity: 66.66%; specificity: 70.83%); (3) ASD and TS+ASD (AUC: 0.78; sensitivity: 73.33%; specificity: 76%). Characterization of miR-140-3p network would contribute to further clarify ASD etiology. Serum miR-140-3p could represent a potential non-invasive biomarker for ASD, easy to test through liquid biopsy. [ABSTRACT FROM AUTHOR]

Age related macular degeneration (AMD) is the leading cause of blindness among people aged 50 and over. Retinal deposition of amyloid-b (Ab) aggregates in AMD patients has suggested a potential link between AMD and Alzheimer's disease (AD). We have evaluated the differential retinal expression profile of miRNAs in a rat model of AMD elicited by Ab. A serum profile of miRNAs in AMD patients has been also assessed using single TaqMan assay. Analysis of retina from rats intravitreally injected with Ab revealed that miR-27a, miR-146a, and miR-155 were up-regulated in comparison to control rats. Seven miRNA (miR-9, miR-23a, miR-27a, miR-34a, miR-126, miR-146a, and miR-155) have been found to be dysregulated in serum of AMD patients in comparison to control group. Analysis of pathways has revealed that dysregulated miRNAs, both in the AMD animal model and in AMD patients, can target genes regulating pathways linked to neurodegeneration and inflammation, reinforcing the hypothesis that AMD is a protein misfolding disease similar to AD. In fact, miR-9, miR-23a, miR-27a, miR-34a, miR-146a, miR-155 have been found to be dysregulated both in AMD and AD. In conclusion, we suggest that miR-9, miR-23a, miR-27a, miR-34a, miR-146a, miR-155 represent potential biomarkers and new pharmacological targets for AMD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vascular dementia (VaD) is a pathogenetically heterogeneous neuropsychiatric syndrome, mainly characterized by cognitive impairment. Among dementias, it is second by incidence after Alzheimer's dementia (AD). VaD biomolecular bases have been poorly characterized, but vascular-linked factors affecting the CNS and its functions are generally hypothesized to perform a major role, together with cardiovascular and immunological factors. miRNAs, which perform critically important biomolecular roles within cell networks, are also found in biological fluids as circulating miRNAs (cmiRNAs). We hypothesized that differentially expressed (DE) cmiRNAs in plasma from VaD patients could be applied to diagnose VaD through liquid biopsies; these profiles also could allow to start investigating VaD molecular bases. By exploiting TaqMan Low-Density Arrays and single TaqMan assays, miR-10b*, miR29a-3p, and miR-130b-3p were discovered and validated as significantly downregulated DE cmiRNAs in VaD patients compared to unaffected controls (NCs). These miRNAs also were found to be significantly downregulated in a matched cohort of AD patients, but miR-130b-3p levels were lower in AD than in VaD. A negative correlation was detected between miR-29a and miR-130b expression and cognitive impairment in VaD and AD, respectively. Receiver operating characteristic curves demonstrated that decreased plasma levels of miR-10b*, miR29a-3p, and miR-130b-3p allow to discriminate VaD and AD patients from NCs. Furthermore, the concurrent downregulation of both miR-10b* and miR-130b-3p in VaD showed an area under the curve (AUC) of 0.789 (p < 0.0001) with 75% of sensitivity and 72% of specificity, whereas an AUC of 0.789 (p < 0.0001) with 92% of sensitivity and 81% of specificity was found for both in AD. The miRNAs profiles reported in this paper pave the way to translational applications to molecular VaD diagnosis, but they also should allow to further investigate on its molecular bases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The study of RNAs in the extracellular environment in physiological and pathological conditions has become a growing field of research with intriguing applications in diagnostics and prognostics. Such extracellular RNAs are passively or actively released by all cells into biological fluids to spread biological signals to other cells. The perturbation of such RNA-based cell-to-cell communications in cancer can be easily identified by molecular analysis of liquid biopsies, even if source cells secreting RNAs are often elusive. In uveal melanoma (UM), extracellular RNAs can be assayed in serum, plasma, and vitreous and aqueous humor. In this review, we explore the possibility that extracellular RNA alterations in UM could partially match with RNA dysregulations observed in tumor tissues and provide information to better understand UM 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. [ABSTRACT FROM AUTHOR]

Male infertility is a risk factor for the development of testicular germ cell tumors. In this study, we investigated microRNA profiles in seminal plasma to identify potential noninvasive biomarkers able to discriminate the men at highest risk of developing cancer among the infertile population. We compared the microRNA profiles of individuals affected by testicular germ cell tumors and healthy individuals with normal or impaired spermiograms using high‐throughput technology and confirmed the results by single‐assay digital PCR. We found that miR‐221‐3p and miR‐222‐3p were downregulated and miR‐126‐3p was upregulated in cancer patients compared to both infertile and fertile men. ROC curve analysis confirmed that miR‐126 upregulation is able to identify cancer patients among the infertile male population. In addition, in‐depth bioinformatics analysis based on weighted gene co‐expression networks showed that the identified miRNAs regulate cellular pathways involved in cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Uveal melanoma (UM) is the most frequent primary tumor of the eye in adults. Although molecular alterations on protein-coding genes have been associated with the development of UM, the role of non-coding RNAs and their competitive endogenous networks remain poorly investigated. Starting from a computational analysis on UM expression dataset deposited in The Cancer Genome Atlas, we identified the long non-coding RNA LINC00518 as a potential oncogene. We then experimentally evaluated LINC00518 and its supposed RNA signaling in human biopsies and in vitro functional assays. The results obtained suggest that LINC00518, under potential transcriptional control by MITF, regulates an RNA–RNA network promoting cancer-related processes (i.e., cell proliferation and migration). These findings open the way to the characterization of the unknown RNA signaling associated with UM and pave the way to the exploitation of a potential target for RNA-based therapeutics. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Circular RNAs are a large group of RNAs whose cellular functions are still being investigated. We recently proposed that circSMARCA5 acts as sponge for the splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1) in glioblastoma multiforme (GBM). After demonstrating by RNA immunoprecipitation a physical interaction between SRFS1 and circSMARCA5, we assayed by real-time PCR in a cohort of 31 GBM biopsies and 20 unaffected brain parenchyma controls (UC) the expression of total, pro-angiogenic (Iso8a) and anti-angiogenic (Iso8b) mRNA isoforms of Vascular Endothelial Growth Factor A (VEGFA), a known splicing target of SRSF1. The Iso8a to Iso8b ratio: (i) increased in GBM biopsies with respect to UC (p-value < 0.00001); (ii) negatively correlated with the expression of circSMARCA5 (r-value = −0.46, p-value = 0.006); (iii) decreased in U87-MG overexpressing circSMARCA5 with respect to negative control (p-value = 0.0055). Blood vascular microvessel density, estimated within the same biopsies, negatively correlated with the expression of circSMARCA5 (r-value = −0.59, p-value = 0.00001), while positively correlated with that of SRSF1 (r-value = 0.38, p-value = 0.00663) and the Iso8a to Iso8b ratio (r-value = 0.41, p-value = 0.0259). Kaplan-Meier survival analysis showed that GBM patients with low circSMARCA5 expression had lower overall and progression free survival rates than those with higher circSMARCA5 expression (p-values = 0.033, 0.012, respectively). Our data convincingly suggest that circSMARCA5 is an upstream regulator of pro- to anti-angiogenic VEGFA isoforms ratio within GBM cells and a highly promising GBM prognostic and prospective anti-angiogenic molecule. [ABSTRACT FROM AUTHOR]

Purpose: Although the expression of microRNAs (miRNAs) in retinal pigment epithelial (RPE) cells undergoing epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of proliferative vitreoretinopathy (PVR), its expression in the vitreous of patients with primary retinal detachment (RD) and different PVR grading has not yet been investigated. We assessed the expression of miRNAs in the vitreous humor (VH) of patients diagnosed with RD and different grading of PVR., Methods: The VH was extracted from the core of the vitreous chamber in patients who had undergone standard vitrectomy for primary RD. RNA was extracted and TaqMan Low-Density Arrays (TLDAs) were used for miRNA profiling that was performed by single TaqMan assays. A gene ontology (GO) analysis was performed on the differentially expressed miRNAs., Results: A total of 15 eyes with RD, 3 eyes for each grade of PVR (A, B, C, and D) and 3 from unaffected individuals, were enrolled in this prospective comparative study. Twenty miRNAs were altered in the comparison among pathological groups. Interestingly, the expression of miR-143-3p, miR-224-5p, miR-361-5p, miR-452-5p, miR-486-3p, and miR-891a-5p increased with the worsening of PVR grading. We also identified 34 miRNAs showing differential expression in PVR compared to control vitreous samples. GO analysis showed that the deregulated miRNAs participate in processes previously associated with PVR pathogenesis., Conclusions: The present pilot study suggested that dysregulated vitreal miRNAs may be considered as a biomarker of PVR and associated with the PVR-related complications in patients with RD., Translational Relevance: The correlation between vitreal miRNAs and the pathological phenotypes are essential to identify the novel miRNA-based mechanisms underlying the PVR disease that would improve the diagnosis and treatment of the condition., Competing Interests: Disclosure: M.D. Toro, None; M. Reibaldi, None; T. Avitabile, None; C. Bucolo, None; S. Salomone, None; R. Rejdak, None; K. Nowomiejska, None; S. Tripodi, None; C. Posarelli, None; M. Ragusa, None; C. Barbagallo, None, (Copyright 2020 The Authors.)

Over the past few years, exosomes and their RNA cargo have been extensively studied because of the fascinating biological roles they play in cell-to-cell communication, including the signal exchange among cancer, stromal, and immune cells, leading to modifications of tumor microenvironment. RNAs, especially miRNAs, stored within exosomes, seem to be among the main determinants of such signaling: their sorting into exosomes appears to be cell-specific and related to cellular physiopathology. Accordingly, the identification of exosomal miRNAs in body fluids from pathological patients has become one of the most promising activity in the field of biomarker discovery. Several analyses on the qualitative and quantitative distribution of RNAs between cells and their secreted exosomes have given rise to questions on whether and how accurately exosomal RNAs would represent the transcriptomic snapshot of the physiological and pathological status of secreting cells. Although the exact molecular mechanisms of sorting remain quite elusive, many papers have reported an evident asymmetric quantitative distribution of RNAs between source cells and their exosomes. This phenomenon could depend both on passive and active sorting mechanisms related to: (a) RNA turnover; (b) maintaining the cytoplasmic miRNA:target equilibrium; (c) removal of RNAs not critical or even detrimental for normal or diseased cells. These observations represent very critical issues in the exploitation of exosomal miRNAs as cancer biomarkers. In this review, we will discuss how much the exosomal and corresponding donor cell transcriptomes match each other, to better understand the actual reliability of exosomal RNA molecules as pathological biomarkers reflecting a diseased status of the cells.

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.

For two decades Vogelstein's model has been the paradigm for describing the sequence of molecular changes within protein-coding genes that would lead to overt colorectal cancer (CRC). This model is now too simplistic in the light of recent studies, which have shown that our genome is pervasively transcribed in RNAs other than mRNAs, denominated non-coding RNAs (ncRNAs). The discovery that mutations in genes encoding these RNAs [i.e., microRNAs (miRNAs), long non-coding RNAs, and circular RNAs] are causally involved in cancer phenotypes has profoundly modified our vision of tumour molecular genetics and pathobiology. By exploiting a wide range of different mechanisms, ncRNAs control fundamental cellular processes, such as proliferation, differentiation, migration, angiogenesis and apoptosis: these data have also confirmed their role as oncogenes or tumor suppressors in cancer development and progression. The existence of a sophisticated RNA-based regulatory system, which dictates the correct functioning of protein-coding networks, has relevant biological and biomedical consequences. Different miRNAs involved in neoplastic and degenerative diseases exhibit potential predictive and prognostic properties. Furthermore, the key roles of ncRNAs make them very attractive targets for innovative therapeutic approaches. Several recent reports have shown that ncRNAs can be secreted by cells into the extracellular environment (i.e., blood and other body fluids): this suggests the existence of extracellular signalling mechanisms, which may be exploited by cells in physiology and pathology. In this review, we will summarize the most relevant issues on the involvement of cellular and extracellular ncRNAs in disease. We will then specifically describe their involvement in CRC pathobiology and their translational applications to CRC diagnosis, prognosis and therapy.

Exchange of molecules via exosomes is a means of eukaryotic intercellular communication, especially within tumour microenvironments. However, no data are available on alterations of exosomal molecular cargo by environmental cues (eg, pharmacological treatments). To approach this issue, we compared the abundance of 754 miRNAs and 741 cancer-related proteins in exosomes secreted by Caco-2 (Cetuximab-responsive) and HCT- 116 (Cetuximab-resistant) CRC cells, before and after Cetuximab treatment, with that in their source cells. Cetuximab significantly altered the cargo of Caco-2 exosomes: it increased abundance of miRNAs and proteins activating proliferation and inflammation and reduced miRNAs and proteins related to immune suppression. These alterations did not precisely mirror those in source cells, suggesting a Cetuximab-linked effect. Analogous alterations were detected in HCT-116. Transfection of exosomes from Cetuximab-treated Caco-2 into HCT-116 significantly increased HCT-116 viability; conversely, no viability alteration was detected in Caco-2 transfected with exosomes from Cetuximab-treated HCT-116. Analysis of networks, comprising targets of differentially expressed (DE) exosomal miRNAs and DE exosomal proteins, demonstrates a significant involvement of processes related to proliferation, inflammation, immune response, apoptosis. Our data extend existing knowledge on molecular mechanisms of eukaryotic intercellular communication, especially in oncological processes. Their translation to clinical settings may add new weapons to existing therapeutic repertoires against cancer.