Davide Corà

The Role of Incoherent MicroRNA-Mediated Feedforward Loops in Noise Buffering

MicroRNAs are endogenous non-coding RNAs which negatively regulate the expression of protein-coding genes in plants and animals. They are known to play an important role in several biological processes and, together with transcription factors, form a complex and highly interconnected regulatory network. Looking at the structure of this network, it is possible to recognize a few overrepresented motifs which are expected to perform important elementary regulatory functions. Among them, a special role is played by the microRNA-mediated feedforward loop in which a master transcription factor regulates a microRNA and, together with it, a set of target genes. In this paper we show analytically and through simulations that the incoherent version of this motif can couple the fine-tuning of a target protein level with an efficient noise control, thus conferring precision and stability to the overall gene expression program, especially in the presence of fluctuations in upstream regulators. Among the other results, a nontrivial prediction of our model is that the optimal attenuation of fluctuations coincides with a modest repression of the target expression. This feature is coherent with the expected fine-tuning function and in agreement with experimental observations of the actual impact of a wide class of microRNAs on the protein output of their targets. Finally, we describe the impact on noise-buffering efficiency of the cross-talk between microRNA targets that can naturally arise if the microRNA-mediated circuit is not considered as isolated, but embedded in a larger network of regulations.

CircuitsDB: a database of mixed microRNA/transcription factor feed-forward regulatory circuits in human and mouse

BackgroundTranscription Factors (TFs) and microRNAs (miRNAs) are key players for gene expression regulation in higher eukaryotes. In the last years, a large amount of bioinformatic studies were devoted to the elucidation of transcriptional and post-transcriptional (mostly miRNA-mediated) regulatory interactions, but little is known about the interplay between them.DescriptionHere we describe a dynamic web-accessible database, CircuitsDB, supporting a genome-wide transcriptional and post-transcriptional regulatory network integration, for the human and mouse genomes, based on a bioinformatic sequence-analysis approach. In particular, CircuitsDB is currently focused on the study of mixed miRNA/TF Feed-Forward regulatory Loops (FFLs), i.e. elementary circuits in which a master TF regulates an miRNA and together with it a set of Joint Target protein-coding genes. The database was constructed using an ab-initio oligo analysis procedure for the identification of the transcriptional and post-transcriptional interactions. Several external sources of information were then pooled together to obtain the functional annotation of the proposed interactions. Results for human and mouse genomes are presented in an integrated web tool, that allows users to explore the circuits, investigate their sequence and functional properties and thus suggest possible biological experiments.ConclusionsWe present CircuitsDB, a web-server devoted to the study of human and mouse mixed miRNA/TF Feed-Forward regulatory circuits, freely available at: http://biocluster.di.unito.it/circuits/

Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations

Purpose: We determined the gene copy numbers for MET, for its transcriptional activator MACC1 and for its ligand hepatocyte growth factor (HGF) in liver metastases from colorectal carcinoma (mCRC). We correlated copy numbers with mRNA levels and explored whether gain and/or overexpression of MET and MACC1 predict response to anti-Met therapies. Finally, we assessed whether their genomic or transcriptional deregulation correlates with pathologic and molecular parameters of aggressive disease. Experimental Design: One hundred three mCRCs were analyzed. Copy numbers and mRNA were determined by quantitative PCR (qPCR). Thirty nine samples were implanted and expanded in NOD (nonobese diabetic)/SCID (severe combined immunodeficient) mice to generate cohorts that were treated with the Met inhibitor JNJ-38877605. In silico analysis of MACC1 targets relied on genome-wide mapping of promoter regions and on expression data from two CRC datasets. Results: No focal, high-grade amplifications of MET, MACC1, or HGF were detected. Chromosome 7 polysomy and gain of the p-arm were observed in 21% and 8% of cases, respectively, and significantly correlated with higher expression of both Met and MACC1. Met inhibition in patient-derived xenografts did not modify tumor growth. Copy number gain and overexpression of MACC1 correlated with unfavorable pathologic features better than overexpression of Met. Bioinformatic analysis of putative MACC1 targets identified elements besides Met, whose overexpression cosegregated with aggressive forms of colorectal cancer. Conclusions: Experiments in patient-derived xenografts suggest that mCRCs do not rely on Met genomic gain and/or overexpression for growth. On the basis of pathologic correlations and bioinformatic analysis, MACC1 could contribute to CRC progression through mechanisms other than or additional to Met transcriptional upregulation. Clin Cancer Res; 17(10); 3146–56. ©2011 AACR.

MicroRNA/gene profiling unveils early molecular changes and nuclear factor erythroid related factor 2 (NRF2) activation in a rat model recapitulating human hepatocellular carcinoma (HCC)

Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then used to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the resistant‐hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin‐19, indicating that several HCC‐associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene‐target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the nuclear factor erythroid related factor 2 (NRF2) pathway and up‐regulation of the miR‐200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR‐200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3‐induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin‐19‐positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated with human HCC as well underlines the translational value of our results. Conclusion: This study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target. (Hepatology 2014;58:228–241)

MicroRNA/gene profiling unveils early molecular changes and NRF2 activation in a rat model recapitulating human HCC.

Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then used to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the resistant‐hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin‐19, indicating that several HCC‐associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene‐target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the nuclear factor erythroid related factor 2 (NRF2) pathway and up‐regulation of the miR‐200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR‐200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3‐induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin‐19‐positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated with human HCC as well underlines the translational value of our results. Conclusion: This study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target. (Hepatology 2014;58:228–241)

Computational identification of transcription factor binding sites by functional analysis of sets of genes sharing overrep-resented upstream motifs

BackgroundTranscriptional regulation is a key mechanism in the functioning of the cell, and is mostly effected through transcription factors binding to specific recognition motifs located upstream of the coding region of the regulated gene. The computational identification of such motifs is made easier by the fact that they often appear several times in the upstream region of the regulated genes, so that the number of occurrences of relevant motifs is often significantly larger than expected by pure chance.ResultsTo exploit this fact, we construct sets of genes characterized by the statistical overrepresentation of a certain motif in their upstream regions. Then we study the functional characterization of these sets by analyzing their annotation to Gene Ontology terms. For the sets showing a statistically significant specific functional characterization, we conjecture that the upstream motif characterizing the set is a binding site for a transcription factor involved in the regulation of the genes in the set.ConclusionsThe method we propose is able to identify many known binding sites in S. cerevisiae and new candidate targets of regulation by known transcritpion factors. Its application to less well studied organisms is likely to be valuable in the exploration of their regulatory interaction network.

Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells

Significance Estrogen receptor-α (ERα) is a key protein in breast cancer and treatments targeting ERα are among the most widely used and effective in clinics. Although the role of estrogen-stimulated ERα in breast cancer has been exhaustively described, the functions of ERα in the absence of estrogen is hill-defined. In this work, we show that ERα binds extensively to the genome of breast cancer cells in the absence of estrogen, where it regulates the expression of hundreds of genes endowed with developmental functions. Our data suggest that ERα has a fundamental role in the homeostasis of luminal epithelial cells also when estrogen is ablated physiologically or pharmacologically. Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.

A Curated Database of miRNA Mediated Feed-Forward Loops Involving MYC as Master Regulator

Background The MYC transcription factors are known to be involved in the biology of many human cancer types. But little is known about the Myc/microRNAs cooperation in the regulation of genes at the transcriptional and post-transcriptional level. Methodology/Principal Findings Employing independent databases with experimentally validated data, we identified several mixed microRNA/Transcription Factor Feed-Forward Loops regulated by Myc and characterized completely by experimentally supported regulatory interactions, in human. We then studied the statistical and functional properties of these circuits and discussed in more detail a few interesting examples involving E2F1, PTEN, RB1 and VEGF. Conclusions/Significance We have assembled and characterized a catalogue of human mixed Transcription Factor/microRNA Feed-Forward Loops, having Myc as master regulator and completely defined by experimentally verified regulatory interactions.

Gene autoregulation via intronic microRNAs and its functions

BackgroundMicroRNAs, post-transcriptional repressors of gene expression, play a pivotal role in gene regulatory networks. They are involved in core cellular processes and their dysregulation is associated to a broad range of human diseases. This paper focus on a minimal microRNA-mediated regulatory circuit, in which a protein-coding gene (host gene) is targeted by a microRNA located inside one of its introns.ResultsAutoregulation via intronic microRNAs is widespread in the human regulatory network, as confirmed by our bioinformatic analysis, and can perform several regulatory tasks despite its simple topology. Our analysis, based on analytical calculations and simulations, indicates that this circuitry alters the dynamics of the host gene expression, can induce complex responses implementing adaptation and Weber’s law, and efficiently filters fluctuations propagating from the upstream network to the host gene. A fine-tuning of the circuit parameters can optimize each of these functions. Interestingly, they are all related to gene expression homeostasis, in agreement with the increasing evidence suggesting a role of microRNA regulation in conferring robustness to biological processes. In addition to model analysis, we present a list of bioinformatically predicted candidate circuits in human for future experimental tests.ConclusionsThe results presented here suggest a potentially relevant functional role for negative self-regulation via intronic microRNAs, in particular as a homeostatic control mechanism of gene expression. Moreover, the map of circuit functions in terms of experimentally measurable parameters, resulting from our analysis, can be a useful guideline for possible applications in synthetic biology.

Molecular and morphologic characterization of superficial- and deep-subcutaneous adipose tissue subdivisions in human obesity

Human abdominal subcutaneous white adipose tissue (SAT) is composed of two different subcompartments: a “superficial” SAT (SSAT), located between the skin and a fibrous‐fascia plane; and a deeper SAT, located under this fibrous fascia plane, indicated as “deep” SAT (DSAT).