MicroRNAs and non-coding RNAs in Development and Disease
The recent discovery of thousands of non-coding RNAs (ncRNAs) with regulatory function is redefining the landscape of transcriptome regulation, highlighting the interplay of epigenetic, transcriptional and post-transcriptional mechanisms in the specification of cell fate and in the regulation of developmental processes. We have witnessed the identification of an increasing number of regulatory small RNAs (such as microRNAs, miRNAs) as well as long non-coding RNAs (lncRNAs) with critical roles in the regulation of molecular circuits associated with numerous biological processes. Fundamentally, all known physiological and pathological processes, including cancer, are regulated by miRNAs and lncRNAs working together to mark differentiation states or acting on their own as authentic oncogenes or tumor suppressors.
Our broad aim is to provide a critical as well original contribution towards the understanding of the role played by ncRNAs in development and disease. We propose an innovative approach i) based on the integration of Experimental models with Computational methodologies, ii) pointing out the reciprocal regulation of coding and non-coding RNAs and iii) combining high-resolution analysis of cancer-specimens and sophisticated models that reproduce cancer alterations.
1. MIR-DECAY: Dynamics of miRNA Regulation in Physiology and Cance
Overall, miRNAs cellullar levels are determined by the sum of two processes: biosynthesis, which generates new miRNA molecules, and decay, that clears old miRNAs. So far, miRNA decay has not been thoroughly investigated in cancer, due to lack of suitable methodologies. In 2016, we developed new tailored approaches based on in vivo RNA labelling and high-throughput sequencing aimed at studying modes and mechanisms of miRNA decay (Marzi, Ghini et al., Genome Res 2016).
Using these methods, we managed to precisely measure miRNA biosynthesis and decay rates in cells, and revealed the existence of different pools of miRNAs with specific decay patterns. Therefore, we showed that, unlike previously thought, not all miRNAs are stable moleules
Despite investigation of miRNA decay mecha-nisms in cancer is still in its early stages, it has recently been shown that targets with extended complementrity can induce miRNA degradation through a mecha-nism known as TDMD, Target-Directed miRNA Degradation. Our lab and others's have recently proved the existence of endogenous TDMD transcripts in mam-malian cells (Ghini, Rubolino et al. Nat Comm 2018), thus establishing the existence of this mechanism in mammalian genomes. To date, the number of en-dogenous TDMD-targets and their possible impact on physiopathology in humans are yet to be defined.
We propose to apply our approaches to models of human cancer to investigate
- the role of decay (and TDMD) in the regulation of miRNAs in cancer
- the critical mechanistic steps associated with this process
2. TIC-LNCRNAs: Long Non-Coding Rnas and Stemness Programme
Despite treatment, some cancers progress by tumor re-initiation, metastasis development or acquisition of therapy resistance. Subpopulations of cancer cells with tumor-initiating capacity and known as cancer stem cells (CSCs) or tumor-initiating cells (TICs) have been implicated in such processes. In breast cancer, in particular, TICs have been shown to sustain tumor re-growth at local (relapse) or distant sites (metastasis) and to contribute to the emergence of therapy resistance. TICs are thought to originate from tumor cells upon activation of a ‘stemness’ programme, a transcriptional programme much similar to that acting in normal tissue on adult stem cells (Bonetti et al. Oncogene 2018, Culurgioni et al. Nat. Comm. 2018).
The ‘stemness’ programme is established and mantained through a series of molecular mechanisms, some of them involving regulatory non-coding RNAs. So far, the role of ncRNAs in this context has been only marginally explored (Tordonato et al. Front. Genet. 2014).
Through the combination of human transcripts high-resolution analysis (high-coverage strand-specific RNA sequencing) and the use of sophisticated biological models reproducing TIC properties, we have managed to isolate a subset of long non coding RNAs (TIC-lncRNAs) extremely relevant as potential novel markers or therapeutic targets for cancer treatment.
Our lab is currently searching for TIC-lncRNAs that are critical for the identity and maintenance of breast TICs with the aim of:
- characterizing underlying molecular mechanisms
- mapping TICs epigenetic and transcriptional landscape in aggressive breast tumors and metastasis
- isolating non-coding elements that functions as determinants of the transcriptional and epigenetic plasticity of cancer cells
3. Epigenetic and Transcriptional Determinants of Cancer Cell Plasticity at Single-Cell Resolution
Different cellular mechanisms acting at epigenetic, transcriptional and post-transcriptional level have been postulated to account for the intrinsic heterogeneity of cancer, this being the main cause of imprecise diagnosis and failure in identifying therapeutic regimens that effectively tackle the disease.
Following recent developments in single cell technologies, our lab is working on producing high-resolution, genome-wide blueprints of the transcriptional and epigenetic mechanisms that regulate plasticity programmes, with the aim of:
- solving the complexity of intrinsic heterogeneity of cancer
- revealing the molecular underpinnings of plasticity (genetic /transcriptional and phenotypic) in tumor cells
- paving the way to the development of new therapies for targeting carcinomas
4. Astrocyte-Mediated Circadian Clock in Neurodegeneration and Brain Ageing - in collaboration with Davide De Pietri Tonelli (IIT - Central Lab)
Animals have an internal timekeeping mechanism that influences cellular metabolic pathways, organ functions and behaviours by precisely regulating circadian rhythms of gene expression. In mammals, the circadian system is centered on the brain and is organized in a hierarchy of multiple oscillators at organ, cellular and molecular level.
Recent studies suggest that astrocytes (the most abundant cell type in the brain) actively participate in the modulation of physiological and circadian behavioral processes in invertebrates (“astroclock”) (Barca-Mayo O. et al. Nat. Comm. 2017).
Based on the hypothesis that the astroclock maintains neural rhythmic behaviour and, in so doing, slows down brain ageing and the associated decline of cognitive functions and peripheral metabolic abnormalities, our lab aims at:
- investigating the molecular and functional mechanisms used by the astroclock
- clarifying the transcriptional and post-transcriptional mechanisms controlling astrocyte-to-neuron communication and the astroclock
- identifying molecular targets valuable as potential new drugs in disorders related with ageing and in age-related brain neuropathology and altered metabolism
IN-HOUSE TOOLS AND METHODOLOGIES
isomiRage: a desktop application that determines the occurrence of different miRNA isoforms based on NGS data, and returns a Bowtie.map alignment file
4sU pulse-chase: 4-Thio-Uridine Metabolic Labelling to measure decay of mature miRNAs
- Matteo J Marzi – Senior Post-doctoral Fellow
- Paola Bonetti – Senior Post-doctoral Fellow
- Montserrat Climent Salarich - Post-doctoral Fellow
- Ines Simeone - Post-doctoral Fellow
- Diego Farinello - Post-doctoral Fellow
- Bianca Giuliani - PhD Student
- Carmela Rubolino - PhD Student
- Claudia Crovace - Scientific Assistant
- Giulia Russo - Undergraduate Student
- Bonetti P, Climent M, Panebianco F, Tordonato C, Santoro A, Marzi MJ, Pelicci PG, Ventura A, Nicassio F. "Dual role from miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer" Oncogene 2018
- Ghini F, Rubolino C, Climent M, Simeone I, Marzi MJ, Nicassio F. "Endogenous Transcripts control miRNA levels and activity in mammalian cells by target-directed miRNA degradation" Nature Communications 2018
- Culurgioni S, Mari S, Bonetti P, Gallini S, Bonetto G, Brennich M, Round A, Nicassio F, Mapelli M. “Insc:LGN tetramers promote asymmetric divisions of mammary stem cells” Nature Communications 2018
- Pons-Espinal M, de Luca E, Marzi MJ, Beckervordersandforth R, Armirotti A, Nicassio F, Fabel K, Kempermann G, De Pietri Tonelli D. “Synergic Functions of miRNAs Determine Neuronal Fate of Adult Neural Stem Cells” Stem Cell Reports 2017
- Marinaro F, Marzi MJ, Hoffmann N, Amin H, Pelizzoli R, Niola F, Nicassio F, De Pietri Tonelli D. “MicroRNA-independent functions of DGCR8 are essential for neocortical development and TBR1 expression” EMBO Reports 2017
- Marzi MJ, Montani F, Carletti RM, Dezi F, Dama E, Bonizzi G, Sandri MT, Rampinelli C, Bellomi M, Maisonneuve P, Spaggiari L, Veronesi G, Bianchi F, Di Fiore PP, Nicassio F. “Optimization and Standardization of Circulating MicroRNA Detection for Clinical Application: The miR-Test Case” Clinical Chemistry 2016
- Marzi MJ, Ghini F, Cerruti B, de Pretis S, Bonetti P, Giacomelli C, Gorski MM, Kress T, Pelizzola M, Muller H, Amati B, Nicassio F. “Degradation dynamics of microRNAs revealed by a novel pulsechase approach” Genome Research 2016