MicroRNAs (miRNA) are small-non coding RNAs endowed with great regulatory power, thus playing key roles not only in almost all physiological pathways, but also in the pathogenesis of several diseases. Surprisingly, genomic distribution analysis revealed the highest density of miRNA sequences on the X chromosome; this evolutionary conserved mammalian feature equips females with a larger miRNA machinery than males. However, miRNAs contribution to some X-related conditions, properties or functions is still poorly explored. With the aim to support and focus research in the field, this review analyzes the literature and databases about X-linked miRNAs, trying to understand how miRNAs could contribute to emerging gender-biased functions and pathological mechanisms, such as immunity and cancer. A fine map of miRNA sequences on the X chromosome is reported, and their known functions are discussed; in addition, bioinformatics functional analyses of the whole X-linked miRNA targetome (predicted and validated) were performed. The emerging scenario points to different gaps in the knowledge that should be filled with future experimental investigations, also in terms of possible implications and pathological perspectives for X chromosome aneuploidy syndromes, such as Turner and Klinefelter syndromes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel RNA virus affecting humans, causing a form of acute pulmonary respiratory disorder named COVID-19, declared a pandemic by the World Health Organization. MicroRNAs (miRNA) play an emerging and important role in the interplay between viruses and host cells. Although the impact of host miRNAs on SARS-CoV-2 infection has been predicted, experimental data are still missing. This study started by a bioinformatics prediction of cellular miRNAs potentially targeting viral RNAs; then, a number of criteria also based on experimental evidence and virus biology were applied, giving rise to eight promising binding miRNAs. Their interaction with viral sequences was experimentally validated by transfecting luciferase-based reporter plasmids carrying viral target sequences or their inverted sequences into the lung A549 cell line. Transfection of the reporter plasmids resulted in a reduction of luciferase activity for five out of the eight potential binding sites, suggesting responsiveness to endogenously expressed miRNAs. Co-transfection of the reporter plasmids along with miRNA mimics led to a further and strong reduction of luciferase activity, validating the interaction between miR-219a-2-3p, miR-30c-5p, miR-378d, miR-29a-3p, miR-15b-5p, and viral sequences. miR-15b was also able to repress plasmid-driven Spike expression. Intriguingly, the viral target sequences are fully conserved in more recent variants such as United Kingdom variant B.1.1.7 and South Africa 501Y.V2. Overall, this study provides a first experimental evidence of the interaction between specific cellular miRNAs and SARS-CoV-2 sequences, thus contributing to understanding the molecular mechanisms underlying virus infection and pathogenesis to envisage innovative therapeutic interventions and diagnostic tools.

Given the increasing interest in bioactive dietary components that can modulate gene expression enhancing human health, three metabolites isolated from hemp seeds—cannabidiolic acid, N-trans-caffeoyltyramine, and cannabisin B—were examined for their ability to change the expression levels of microRNAs in human neural cells. To this end, cultured SH-SY5Y cells were treated with the three compounds and their microRNA content was characterized by next-generation small RNA sequencing. As a result, 31 microRNAs underwent major expression changes, being at least doubled or halved by the treatments. A computational analysis of the biological pathways affected by these microRNAs then showed that some are implicated in neural functions, such as axon guidance, hippocampal signaling, and neurotrophin signaling. Of these, miR-708-5p, miR-181a-5p, miR-190a-5p, miR-199a-5p, and miR-143-3p are known to be involved in Alzheimer’s disease and their expression changes are expected to ameliorate neural function. Overall, these results provide new insights into the mechanism of action of hemp seed metabolites and encourage further studies to gain a better understanding of their biological effects on the central nervous system.

MicroRNAs (miRNA), and more recently long non-coding RNAs (lncRNA), are emerging as a driving force for hepatocellular carcinoma (HCC), one of the leading causes of cancer-related death. In this work, we investigated a possible RNA regulatory network involving two oncosuppressive miRNAs, miR-125a and let-7e, and a long non-coding antisense RNA, SPACA6P-AS (SP-AS), all transcribed from the same locus, with SP-AS in the opposite direction and thus carrying complementary sequences to the miRNAs. In vitro experiments validated the binding of the miRNAs to SP-AS. Then, the boosting of either the miRNAs or SP-AS levels demonstrated their reciprocal inhibition. In addition, overexpression of SP-AS resulted in a reduced silencing activity of miR-125a and let-7e toward their key oncogenic targets, i.e., Lin28b, MMP11, SIRT7, Zbtb7a, Cyclin D1, CDC25B, HMGA2, that resulted significantly upregulated. Finally, the analysis of 374 HCC samples in comparison to 50 normal liver tissues showed an upregulation of SP-AS and a reverse expression of miR-125a, not observed for let-7e; consistently, miR-125a oncogenic targets were upregulated. Overall, the data depict a novel competing endogenous RNA (ceRNA) network, ceRNET, whereby miR-125a can regulate the expression of SP-AS, which in turn regulates the miRNA by competing with the binding to the mRNA targets. We speculate that the unbalancing of any network component may contribute to hepatocarcinogenesis.

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CircRNA cargo in spermatozoa (SPZ) participates in setting cell quality, in terms of morphology and motility. Cannabinoid receptor CB1 activity is correlated with a proper spermatogenesis and epididymal sperm maturation. Despite CB1 promotes endogenous skill to circularize mRNAs in SPZ, few notions are reported regarding the functional link between endocannabinoids and spermatic circRNA cargo. In CB1 knock-out male mice, we performed a complete dataset of spermatic circRNA content by microarray strategy. Differentially expressed (DE)-circRNAs, as a function of genotype, were identified. Within DE-circRNAs, we focused the attention on circLIMA1, as putative actin-cytoskeleton architecture regulator. The validation of circLIMA1 dependent-competitive endogenous RNA (ceRNA) network (ceRNET) in in vitro cell line confirmed its activity in the regulation of the cytoskeletal actin. Interestingly, a dynamic actin regulation in SPZ nuclei was found during their epididymal maturation. In this scenario, we showed for the first time an intriguing sperm nuclear actin remodeling, regulated via a ceRNET-independent pathway, consisting in the nuclear shuttling of circLIMA1-QKI interactome and downstream in Gelsolin regulation. In particular, the increased levels of circLIMA1 in CB1 knock-out SPZ, associated with an inefficient depolymerization of nuclear actin, specifically illustrate how endocannabinoids, by regulating circRNA cargo, may contribute to sperm morpho-cellular maturation.

Non-coding RNAs (ncRNAs) constitute the majority of the transcriptome, as the result of pervasive transcription of the mammalian genome. Different RNA species, such as lncRNAs, miRNAs, circRNA, mRNAs, engage in regulatory networks based on their reciprocal interactions, often in a competitive manner, in a way denominated “competing endogenous RNA (ceRNA) networks” (“ceRNET”): miRNAs and other ncRNAs modulate each other, since miRNAs can regulate the expression of lncRNAs, which in turn regulate miRNAs, titrating their availability and thus competing with the binding to other RNA targets. The unbalancing of any network component can derail the entire regulatory circuit acting as a driving force for human diseases, thus assigning “new” functions to “old” molecules. This is the case of XIST, the lncRNA characterized in the early 1990s and well known as the essential molecule for X chromosome inactivation in mammalian females, thus preventing an imbalance of X-linked gene expression between females and males. Currently, literature concerning XIST biology is becoming dominated by miRNA associations and they are also gaining prominence for other lncRNAs produced by the X-inactivation center. This review discusses the available literature to explore possible novel functions related to ceRNA activity of lncRNAs produced by the X-inactivation center, beyond their role in dosage compensation, with prospective implications for emerging gender-biased functions and pathological mechanisms.

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