Unveil Intrahepatic Cholangiocarcinoma Heterogeneity through the Lens of Omics and Multi-Omics Approaches.

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.

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.

Genetics and RNA Regulation of Uveal Melanoma.

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.

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.

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.

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.

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.

Circulating microRNAs Profile in Patients With Transthyretin Variant Amyloidosis.

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.

LINC00483 Has a Potential Tumor-Suppressor Role in Colorectal Cancer Through Multiple Molecular Axes.

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 TGFß-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.

CircSMARCA5 Regulates VEGFA mRNA Splicing and Angiogenesis in Glioblastoma Multiforme Through the Binding of SRSF1.

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.

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

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

LncRNA UCA1, Upregulated in CRC Biopsies and Downregulated in Serum Exosomes, Controls mRNA Expression by RNA-RNA Interactions.

Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) contribute to the onset of many neoplasias through RNA-RNA competitive interactions; in addition, they could be secreted by cancer cells into biological fluids, suggesting their potential diagnostic application. By analyzing the expression of 17 lncRNAs and 31 circRNAs in biopsies and serum exosomes from colorectal cancer (CRC) patients through qRT-PCR, we detected CCAT1, CCAT2, HOTAIR, and UCA1 upregulation and CDR1AS, MALAT1, and TUG1 downregulation in biopsies. In serum exosomes, UCA1 was downregulated, while circHIPK3 and TUG1 were upregulated. Combined receiver operating characteristic (ROC) curves of TUG1:UCA1 and circHIPK3:UCA1 showed high values of sensitivity and specificity. Through in vitro (i.e., RNA silencing and mitogen-activated protein kinase [MAPK] inhibition) and in silico analyses (i.e., expression correlation and RNA-RNA-binding prediction), we found that UCA1 could (1) be controlled by MAPKs through CEBPB; (2) sequester miR-135a, miR-143, miR-214, and miR-1271, protecting ANLN, BIRC5, IPO7, KIF2A, and KIF23 from microRNA (miRNA)-induced degradation; and (3) interact with mRNA 3'-UTRs, preventing miRNA binding. UCA1 and its co-regulated antisense LINC01764 could interact and reciprocally mask their own miRNA-binding sites. Functional enrichment analysis of the RNA-RNA network controlled by UCA1 suggested its potential involvement in cellular migration. The UCA1 regulatory axis would represent a promising target to develop innovative RNA-based therapeutics against CRC.

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

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

Molecular Crosstalking among Noncoding RNAs: A New Network Layer of Genome Regulation in Cancer.

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.

Asymmetric RNA Distribution among Cells and Their Secreted Exosomes: Biomedical Meaning and Considerations on Diagnostic Applications.

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.

Expression and Regulatory Network Analysis of miR-140-3p, a New Potential Serum Biomarker for Autism Spectrum Disorder.

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). ΔCt 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.

Altered expression of miRNAs and methylation of their promoters are correlated in neuroblastoma.

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.

Non-coding landscapes of colorectal cancer.

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.