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Cancer is a highly heterogeneous disease, where phenotypically distinct subpopulations coexist and can be primed to different fates. Both genetic and epigenetic factors may drive cancer evolution, however little is known about whether and how such a process is pre-encoded in cancer clones. Using single-cell multi-omic lineage tracing and phenotypic assays, we investigate the predictive features of either tumour initiation or drug tolerance within the same cancer population. Clones primed to tumour initiation in vivo display two distinct transcriptional states at baseline. Remarkably, these states share a distinctive DNA accessibility profile, highlighting an epigenetic basis for tumour initiation. The drug tolerant niche is also largely pre-encoded, but only partially overlaps the tumour-initiating one and evolves following two genetically and transcriptionally distinct trajectories. Our study highlights coexisting genetic, epigenetic and transcriptional determinants of cancer evolution, unravelling the molecular complexity of pre-encoded tumour phenotypes., (© 2024. The Author(s).)

The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

It is currently unknown how many RNA transcripts are able to induce degradation of microRNAs (miRNA) via the mechanism known as target-directed miRNA degradation (TDMD). We developed TDMDfinder, a computational pipeline that identifies 'high confidence' TDMD interactions in the Human and Mouse transcriptomes by combining sequence alignment and feature selection approaches. Our predictions suggested that TDMD is widespread, with potentially every miRNA controlled by endogenous targets. We experimentally tested 37 TDMDfinder predictions, of which 17 showed TDMD effects as measured by RT-qPCR and small RNA sequencing, linking the miR-17, miR-19, miR-30, miR-221, miR-26 and miR-23 families to novel endogenous TDMDs. In some cases, TDMD was found to affect different members of the same miRNA family selectively. Features like complementarity to the miRNA 3' region, bulge size and hybridization energy appeared to be the main factors determining sensitivity. Computational analyses performed using the multiomic TCGA platform substantiated the involvement of many TDMD transcripts in human cancer and highlighted 36 highly significant interactions, suggesting TDMD as a new potential oncogenic mechanism. In conclusion, TDMDfinder provides the first inventory of bona fide human and mouse TDMDs. Available as a free webtool, TDMDfinder allows users to search for any TDMD interaction of interest by customizing its selection criteria., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MicroRNAs (miRNAs) are an evolutionarily conserved class of small (18-22 nucleotides) noncoding RNAs involved in the regulation of a variety of cellular and developmental processes. MiRNA expression is frequently altered in human cancers compared to normal tissues, potentially contributing to tumorigenesis. Generally, high-throughput profiles of miRNA expression levels are generated using bulk samples, from both normal and cancer tissues. However, cancer tissues are quite heterogeneous and might contain subpopulations critical for tumor development, i.e., cancer stem cells (CSCs) or tumor-initiating cells (TICs) with aberrant stem-like features, such as unlimited self-renewal potential. The isolation of these aberrant subpopulations from solid tumors is a relatively recent achievement, with breast cancer being one of the first solid human cancers in which CSCs have been identified and biologically characterized. Here, we describe a new methodology that can overcome the main challenge in dealing with rare cells such as SCs/CSCs, represented by the paucity of the starting material. Based on previously published protocols, used by both our and other research groups, we used the FACS-sorting approach to isolate mammary normal and cancer stem cells based on the amount of PKH26 fluorescent dye they retained. Depending on the number of SCs/CSCs isolated, we established two different protocols for the reliable and analytically sensitive detection of up to 384 miRNAs using the Taqman Low Density Array (TLDA) platform., Competing Interests: Disclosures C.T., M.J.M., P.P.D.F. and F.N. have no conflicts of interest to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Recent findings in human samples and animal models support the involvement of inflammation in the development of Parkinson's disease. Nevertheless, it is currently unknown whether microglial activation constitutes a primary event in neurodegeneration. We generated a new mouse model by lentiviral-mediated selective α-synuclein (αSYN) accumulation in microglial cells. Surprisingly, these mice developed progressive degeneration of dopaminergic (DA) neurons without endogenous αSYN aggregation. Transcriptomics and functional assessment revealed that αSYN-accumulating microglial cells developed a strong reactive state with phagocytic exhaustion and excessive production of oxidative and proinflammatory molecules. This inflammatory state created a molecular feed-forward vicious cycle between microglia and IFNγ-secreting immune cells infiltrating the brain parenchyma. Pharmacological inhibition of oxidative and nitrosative molecule production was sufficient to attenuate neurodegeneration. These results suggest that αSYN accumulation in microglia induces selective DA neuronal degeneration by promoting phagocytic exhaustion, an excessively toxic environment and the selective recruitment of peripheral immune cells., (© 2021. The Author(s).)

Although ectopic overexpression of miRNAs can influence mammary normal and cancer stem cells (SCs/CSCs), their physiological relevance remains uncertain. Here, we show that miR-146 is relevant for SC/CSC activity. MiR-146a/b expression is high in SCs/CSCs from human/mouse primary mammary tissues, correlates with the basal-like breast cancer subtype, which typically has a high CSC content, and specifically distinguishes cells with SC/CSC identity. Loss of miR-146 reduces SC/CSC self-renewal in vitro and compromises patient-derived xenograft tumor growth in vivo, decreasing the number of tumor-initiating cells, thus supporting its pro-oncogenic function. Transcriptional analysis in mammary SC-like cells revealed that miR-146 has pleiotropic effects, reducing adaptive response mechanisms and activating the exit from quiescent state, through a complex network of finely regulated miRNA targets related to quiescence, transcription, and one-carbon pool metabolism. Consistent with these findings, SCs/CSCs display innate resistance to anti-folate chemotherapies either in vitro or in vivo that can be reversed by miR-146 depletion, unmasking a "hidden vulnerability" exploitable for the development of anti-CSC therapies., (© 2021 Tordonato et al.)

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

Physical exercise stimulates adult hippocampal neurogenesis and is considered a relevant strategy for preventing age-related cognitive decline in humans. The underlying mechanisms remains controversial. Here, we show that exercise increases proliferation of neural precursor cells (NPCs) of the mouse dentate gyrus (DG) via downregulation of microRNA 135a-5p (miR-135a). MiR-135a inhibition stimulates NPC proliferation leading to increased neurogenesis, but not astrogliogenesis, in DG of resting mice, and intriguingly it re-activates NPC proliferation in aged mice. We identify 17 proteins (11 putative targets) modulated by miR-135 in NPCs. Of note, inositol 1,4,5-trisphosphate (IP3) receptor 1 and inositol polyphosphate-4-phosphatase type I are among the modulated proteins, suggesting that IP3 signaling may act downstream miR-135. miR-135 is the first noncoding RNA essential modulator of the brain's response to physical exercise. Prospectively, the miR-135-IP3 axis might represent a novel target of therapeutic intervention to prevent pathological brain aging., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-β and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-β.

The role of the tumour-suppressor miR-34 family in breast physiology and in mammary stem cells (MaSCs) is largely unknown. Here, we revealed that miR-34 family, and miR-34a in particular, is implicated in mammary epithelium homoeostasis. Expression of miR-34a occurs upon luminal commitment and differentiation and serves to inhibit the expansion of the pool of MaSCs and early progenitor cells, likely in a p53-independent fashion. Mutant mice (miR34-KO) and loss-of-function approaches revealed two separate functions of miR-34a, controlling both proliferation and fate commitment in mammary progenitors by modulating several pathways involved in epithelial cell plasticity and luminal-to-basal conversion. In particular, miR-34a acts as endogenous inhibitor of the Wnt/beta-catenin signalling pathway, targeting up to nine upstream regulators at the same time, thus modulating the expansion of the MaSCs/early progenitor pool. These multiple roles of miR-34a are maintained in a model of human breast cancer, in which chronic expression of miR-34a in triple-negative mesenchymal-like cells (enriched in cancer stem cells-CSCs) could promote a luminal-like differentiation programme, restrict the CSC pool, and inhibit tumour propagation. Hence, activation of miR-34a-dependent programmes could provide a therapeutic opportunity for the subset of breast cancers, which are rich in CSCs and respond poorly to conventional therapies.

Little is known about miRNA decay. A target-directed miRNA degradation mechanism (TDMD) has been suggested, but further investigation on endogenous targets is necessary. Here, we identify hundreds of targets eligible for TDMD and show that an endogenous RNA (Serpine1) controls the degradation of two miRNAs (miR-30b-5p and miR-30c-5p) in mouse fibroblasts. In our study, TDMD occurs when the target is expressed at relatively low levels, similar in range to those of its miRNAs (100-200 copies per cell), and becomes more effective at high target:miRNA ratios (>10:1). We employ CRISPR/Cas9 to delete the miR-30 responsive element within Serpine1 3'UTR and interfere with TDMD. TDMD suppression increases miR-30b/c levels and boosts their activity towards other targets, modulating gene expression and cellular phenotypes (i.e., cell cycle re-entry and apoptosis). In conclusion, a sophisticated regulatory layer of miRNA and gene expression mediated by specific endogenous targets exists in mammalian cells.

MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (<18-22 nt in length) that act in posttranscriptional regulation of gene expression in higher eukaryotes. The abundance of a miRNA is a key feature in control of its activity and, therefore, a number of mechanisms finely regulate miRNA levels, acting at both transcriptional and posttranscriptional level. Recent evidences, including our research, highlighted the role of miRNA decay as a mechanism controlling the miRNA pool. We describe in this chapter an optimized methodology to determine miRNA degradation rates in mammalian cells. Our approach is based on metabolic pulse labeling with 4-thiouridine (4sU), a uridine analog that is incorporated in nascent RNA and allows thiol-specific biotinylation and selective pull-down of labeled RNA. In particular, given the long average half-life and the complex biogenetic process of miRNAs, we developed a "pulse-chase" protocol where 4sU is removed from the medium after a long labeling period (2-3 h pulse), and labeled RNA is purified at different time points to measure the decay of labeled molecules. By combining the 4sU-based "pulse-chase" approach with high-throughput small RNA sequencing (sRNAseq), it is possible to quantify at genome-wide level miRNA degradation rates.

Adult neurogenesis requires the precise control of neuronal versus astrocyte lineage determination in neural stem cells. While microRNAs (miRNAs) are critically involved in this step during development, their actions in adult hippocampal neural stem cells (aNSCs) has been unclear. As entry point to address that question we chose DICER, an endoribonuclease essential for miRNA biogenesis and other RNAi-related processes. By specific ablation of Dicer in aNSCs in vivo and in vitro, we demonstrate that miRNAs are required for the generation of new neurons, but not astrocytes, in the adult murine hippocampus. Moreover, we identify 11 miRNAs, of which 9 have not been previously characterized in neurogenesis, that determine neurogenic lineage fate choice of aNSCs at the expense of astrogliogenesis. Finally, we propose that the 11 miRNAs sustain adult hippocampal neurogenesis through synergistic modulation of 26 putative targets from different pathways., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent evidence indicates that the miRNA biogenesis factors DROSHA, DGCR8, and DICER exert non-overlapping functions, and have also roles in miRNA-independent regulatory mechanisms. However, it is currently unknown whether miRNA-independent functions of DGCR8 play any role in the maintenance of neuronal progenitors and during corticogenesis. Here, by phenotypic comparison of cortices from conditional Dgcr8 and Dicer knockout mice, we show that Dgcr8 deletion, in contrast to Dicer depletion, leads to premature differentiation of neural progenitor cells and overproduction of TBR1-positive neurons. Remarkably, depletion of miRNAs upon DCGR8 loss is reduced compared to DICER loss, indicating that these phenotypic differences are mediated by miRNA-independent functions of DGCR8. We show that Dgcr8 mutations induce an earlier and stronger phenotype in the developing nervous system compared to Dicer mutants and that miRNA-independent functions of DGCR8 are critical for corticogenesis. Finally, our data also suggest that the Microprocessor complex, with DROSHA and DGCR8 as core components, directly regulates the Tbr1 transcript, containing evolutionarily conserved hairpins that resemble miRNA precursors, independently of miRNAs., (© 2017 The Authors.)

Background: The identification of circulating microRNAs (miRNAs) in the blood has been recently exploited for the development of minimally invasive tests for the early detection of cancer. Nevertheless, the clinical transferability of such tests is uncertain due to still-insufficient standardization and optimization of methods to detect circulating miRNAs in the clinical setting., Methods: We performed a series of tests to optimize the quantification of serum miRNAs that compose the miR-Test, a signature for lung cancer early detection, and systematically analyzed variables that could affect the performance of the test. We took advantage of a large-scale (>1000 samples) validation study of the miR-Test that we recently published, to evaluate, in clinical samples, the effects of analytical and preanalytical variables on the quantification of circulating miRNAs and the clinical output of the signature (risk score)., Results: We developed a streamlined and standardized pipeline for the processing of clinical serum samples that allows the isolation and analysis of circulating miRNAs by quantitative reverse-transcription PCR, with a throughput compatible with screening trials. The major source of analytical variation came from RNA isolation from serum, which could be corrected by use of external (spike-in) or endogenous miRNAs as a reference for normalization. We also introduced standard operating procedures and QC steps to check for unspecific fluctuations that arise from the lack of standardized criteria in the collection or handling of the samples (preanalytical factors)., Conclusions: We propose our methodology as a reference for the development of clinical-grade blood tests on the basis of miRNA detection., (© 2016 American Association for Clinical Chemistry.)

The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions., (© 2016 Marzi et al.; Published by Cold Spring Harbor Laboratory Press.)

As more small RNA sequencing libraries are becoming available, it clearly emerges that microRNAs (miRNAs) are highly heterogeneous both in length and sequence. In comparison to canonical miRNAs, miRNA isoforms (termed as "isomiRs") might exhibit different biological properties, such as a different target repertoire, or enhanced/reduced stability. Nonetheless, this layer of information has remained largely unexplored due to the scarcity of small RNA NGS-datasets and the absence of proper analytical tools. Here, we present a workflow for the characterization and analysis of miRNAs and their variants in next-generation sequencing datasets. IsomiRs can originate from an alternative dicing event ("templated" forms) or from the addition of nucleotides through an enzymatic activity or target-dependent mechanisms ("non-templated" forms). Our pipeline allows distinguishing canonical miRNAs from templated and non-templated isomiRs by alignment to a custom database, which comprises all possible 3'-, 5'-, and trimmed variants. Functionally equivalent isomiRs can be grouped together according to the type of modification (e.g., uridylation, adenylation, trimming …) to assess which miRNAs are more intensively modified in a given biological context. When applied to the analysis of primary epithelial breast cancer cells, our methodology provided a 40% increase in the number of detected miRNA species and allowed to easily identify and classify more than 1000 variants. Most modifications were compatible with templated IsomiRs, as a consequence of imprecise Drosha or Dicer cleavage. However, some non-templated variants were consistently found either in the normal or in the cancer cells, with the 3'-end adenylation and uridylation as the most frequent events, suggesting that miRNA post-transcriptional modification frequently occurs. In conclusion, our analytical tool permits the deconvolution of miRNA heterogeneity and could be used to explore the functional role of miRNA isoforms.

The cancer-associated loss of microRNA (miRNA) expression leads to a proliferative advantage and aggressive behavior through largely unknown mechanisms. Here, we exploit a model system that recapitulates physiological terminal differentiation and its reversal upon oncogene expression to analyze coordinated mRNA/miRNA responses. The cell cycle reentry of myotubes, forced by the E1A oncogene, was associated with a pattern of mRNA/miRNA modulation that was largely reciprocal to that induced during the differentiation of myoblasts into myotubes. The E1A-induced mRNA response was preponderantly Retinoblastoma protein (Rb)-dependent. Conversely, the miRNA response was mostly Rb-independent and exerted through tissue-specific factors and Myc. A subset of these miRNAs (miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7) was shown to coordinately target Rb-dependent cell cycle and DNA replication mRNAs. Thus, a dual level of regulation-transcriptional regulation via Rb-E2F and posttranscriptional regulation via miRNAs-confers robustness to cell cycle control and provides a molecular basis to understand the role of miRNA subversion in cancer.