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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.

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.

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., Graphical Abstract

Circular RNAs (circRNAs) have a critical role in the control of gene expression. Their function in spermatozoa (SPZ) is unknown to date. Twenty-eight genes, involved in SPZ/testicular and epididymal physiology, were given in circBase database to find which of them may generate circular transcripts. We focused on circNAPEPLDiso1, one of the circular RNA isoforms of NAPEPLD transcript, because expressed in human and murine SPZ. In order to functionally characterize circNAPEPLDiso1 as potential microRNA (miRNA) sponge, we performed circNAPEPLDiso1-miR-CATCH and then profiled the expression of 754 miRNAs, by using TaqMan (R) Low Density Arrays. Among them, miRNAs 146a-5p, 203a-3p, 302c-3p, 766-3p and 1260a (some of them previously shown to be expressed in the oocyte), resulted enriched in circNAPEPLDiso1-miR-CATCHed cell lysate: the network of interactions generated from their validated targets was centred on a core of genes involved in the control of cell cycle. Moreover, computational analysis of circNAPEPLDiso1 sequence also showed its potential translation in a short form of NAPEPLD protein. Interestingly, the expression analysis in murine-unfertilized oocytes revealed low and high levels of circNAPEPLDiso1 and circNAPEPLDiso2, respectively. After fertilization, circNAPEPLDiso1 expression significantly increased, instead circNAPEPLDiso2 expression appeared constant. Based on these data, we suggest that SPZ-derived circNAPEPLDiso1 physically interacts with miRNAs primarily involved in the control of cell cycle; we hypothesize that it may represent a paternal cytoplasmic contribution to the zygote and function as a miRNA decoy inside the fertilized oocytes to regulate the first stages of embryo development. This role is proposed here for the first time.

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.

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.