Small RNA profiling reveals deregulated phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/Akt pathway in bronchial smooth muscle cells from asthmatic patients.

BACKGROUND: Aberrant expression of small noncoding RNAs (sncRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) in particular, define several pathologic processes. Asthma is characterized by airway hyperreactivity, chronic inflammation, and airway wall remodeling. Asthma-specific miRNA profiles were reported for bronchial epithelial cells, whereas sncRNA expression in asthmatic bronchial smooth muscle (BSM) cells is almost completely unexplored. OBJECTIVE: We sought to determine whether the primary BSM sncRNA expression profile is altered in asthmatic patients and identify targets of differentially expressed sncRNAs. METHODS: Small RNA sequencing was used for sncRNA profiling in BSM cells (from 8 asthmatic and 6 nonasthmatic subjects). sncRNA identification and differential expression analysis was performed with iMir software. Experimentally validated miRNA targets were identified by using Ingenuity Pathway Analysis, and putative piRNA targets were identified by using miRanda software. RESULTS: BSM cells from asthmatic patients showed abnormal expression of 32 sncRNAs (26 miRNAs, 5 piRNAs, and 1 small nucleolar RNA). Target prediction for deregulated miRNAs and piRNAs revealed experimentally validated and predicted mRNA targets expressed in the BSM cells. Thirty-eight of these mRNAs represent major targets for deregulated miRNAs and might play important roles in the pathophysiology of asthma. Interestingly, 6 of these mRNAs were previously associated with asthma, considered as novel therapeutic targets for treatment of this disease, or both. Signaling pathway analysis revealed involvement of 38 miRNA-targeted mRNAs in increased cell proliferation through phosphatase and tensin homolog and phosphoinositide 3-kinase/Akt signaling pathways. CONCLUSIONS: BSM cells of asthmatic patients are characterized by aberrant sncRNA expression that recapitulates multiple pathologic phenotypes of these cells.

The "busy life" of unliganded estrogen receptors.

Understanding of the role of estrogen receptors (ERα and ERß) in the pathophysiology of breast cancer (BC) has considerably increased in last decades. Despite sharing a similar structure, these two transcription factors often exert opposite roles in BC. In addition, it has been shown that their transcriptional activity is not strictly associated to ligand activation and that unliganded ERs are able to "have a life on their own." This appears to be mainly due to ligand-independent mechanisms leading to ERs PTMs or to their recruitment to specific protein complexes, dependent on cellular context. Furthermore, a significant unliganded ER activity, probably independent by the activation of other pathways, has been recently reported to affect gene transcription, microRNA expression, and downstream proteome. In this review, we describe recent findings on nuclear and cytoplasmic unliganded ERα and ERß activity. We focus on functional genomics, epigenomics, and interaction proteomics data, including PTM induced by ERs-modulated miRNAs in the BC context. A better comprehension of the molecular events controlled by unliganded ERs activity in BC pathogenesis is crucial since it may impact the therapeutic approach to the initial or acquired resistance to endocrine therapies, frequently experienced in the treatment of BC.

Post-transcriptional regulation of human breast cancer cell proteome by unliganded estrogen receptor ß via microRNAs.

Estrogen receptor ß (ERß) is a member of the nuclear receptor family of homeostatic regulators that is frequently lost in breast cancer (BC), where its presence correlates with a better prognosis and a less aggressive clinical outcome of the disease. In contrast to ERα, its closest homolog, ERß shows significant estrogen-independent activities, including the ability to inhibit cell cycle progression and regulate gene transcription in the absence of the ligand. Investigating the nature and extent of this constitutive activity of ERß in BC MCF-7 and ZR-75.1 cells by means of microRNA (miRNA) sequencing, we identified 30 miRNAs differentially expressed in ERß+ versus ERß- cells in the absence of ligand, including up-regulated oncosuppressor miRs such miR-30a. In addition, a significant fraction of >1,600 unique proteins identified in MCF-7 cells by iTRAQ quantitative proteomics were either increased or decreased by ERß, revealing regulation of multiple cell pathways by ligand-free receptors. Transcriptome analysis showed that for a large number of proteins regulated by ERß, the corresponding mRNAs are unaffected, including a large number of putative targets of ERß-regulated miRNAs, indicating a central role of miRNAs in mediating BC cell proteome regulation by ERß. Expression of a mimic of miR-30a-5p, a direct target and downstream effector of ERß in BC, led to the identification of several target transcripts of this miRNA, including 11 encoding proteins whose intracellular concentration was significantly affected by unliganded receptor. These results demonstrate a significant effect of ligand-free ERß on BC cell functions via modulation of the cell proteome and suggest that miRNA regulation might represent a key event in the control of the biological and clinical phenotype of hormone-responsive BC by this nuclear receptor.

Identification of cytoplasmic proteins interacting with unliganded estrogen receptor α and ß in human breast cancer cells.

Estrogen receptor subtypes (ERα and ERß) are transcription factors sharing a similar structure but exerting opposite roles in breast cancer cells. Besides the well-characterized genomic actions of nuclear ERs upon ligand binding, specific actions of ligand-free ERs in the cytoplasm also affect cellular functions. The identification of cytoplasmic interaction partners of unliganded ERα and ERß may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, cytoplasmic extracts from human breast cancer MCF-7 cells stably expressing tandem affinity purification-tagged ERα and ERß and maintained in estrogen-free medium were subject to affinity-purification and MS analysis, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERß, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways targeted by each receptor in the cytoplasm. This work, reporting the first identification of the unliganded ERα and ERß cytoplasmic interactomes in breast cancer cells, provides novel experimental evidence on the nongenomic effects of ERs in the absence of hormonal stimulus. All MS data have been deposited in the ProteomeXchange with identifier PXD001202 (http://proteomecentral.proteomexchange.org/dataset/PXD001202).

Effect of low frequency (LF) electric fields on gene expression of a bone human cell line.

We evaluated the effects, on cultured human SaOS-2 cells, of exposures to the low frequency (LF) electric signal (60 kHz sinusoidal wave, 24.5 V peak-to-peak voltage, amplitude modulated by a 12.5 Hz square wave, 50% duty cycle) from an apparatus of current clinical use in bone diseases requiring regenerating processes. Cells in flasks were exposed to a capacitively coupled electric field giving electric current density in the sample of 4 µA/cm(2). The whole expressed cellular mRNAs were systematically analyzed by "DNA microchips" technology to identify all individual species quantitatively affected by field exposure. Comparisons were made between RNA samples from exposed and control sham-exposed cells. Results indicated that immediately and 4 h after exposure there were almost no differentially modulated mRNA species. However, samples obtained at 24 h after exposure showed a small number of limitedly differential signals (7 down-regulated and 3 up-regulated with a cut-off value of ±1.5; 38 and 11, respectively, with a cut-off value of ±1.3), which included mostly mRNA encoding transcription factors and DNA binding proteins. Nevertheless, in identical experimental conditions, we previously demonstrated enzymatic changes of alkaline phosphatase occurring immediately after exposure and declining in a few hours. Therefore, since enzymatic changes occur before those observed at gene regulation level, it is conceivable that only earlier effects are directly due the treatment and then these effects are later able to affect gene expression only indirectly.

Molecular mechanisms of selective estrogen receptor modulator activity in human breast cancer cells: identification of novel nuclear cofactors of antiestrogen-ERα complexes by interaction proteomics.

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways via protein-protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17ß-estradiol/E(2)), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein-protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E(2))- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen-ERα complexes in human BC cell nuclei. In particular, the E(2)-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds.

Smarcad1 mediates microbiota-induced inflammation in mouse and coordinates gene expression in the intestinal epithelium.

BACKGROUND: How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue. RESULTS: Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum. CONCLUSIONS: Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases.

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells.

BACKGROUND: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. RESULTS: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. CONCLUSIONS: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.

Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases.

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.

Small non-coding RNA deregulation in endometrial carcinogenesis.

Small non-coding RNAs (sncRNAs) represent a heterogeneous group of <200nt-long transcripts comprising microRNAs, PIWI-interacting RNAs (piRNAs) and small-nucleolar-RNAs (snoRNAs) involved in physiological and pathological processes such as carcinogenesis and tumor progression. Aberrant sncRNA expression in cancer has been associated with specific clinical phenotypes, grading, staging, metastases development and resistance to therapy.Aim of the present work is to study the role of sncRNAs in endometrial carcinogenesis. Changes in sncRNA expression were identified by high-throughput genomic analysis of paired normal, hyperplastic and cancerous endometrial tissues obtained by endometrial biopsies (n = 10). Using smallRNA sequencing and microarrays we identified significant differences in sncRNA expression pattern between normal, hyperplastic and neoplastic endometrium. This led to the definition of a sncRNA signature (129 microRNAs, 2 of which not previously described, 10 piRNAs and 3 snoRNAs) of neoplastic transformation. Functional bioinformatics analysis identified as downstream targets multiple signaling pathways potentially involved in the hyperplastic and neoplastic tissue responses, including Wnt/ß-catenin, and ERK/MAPK and TGF-ß-Signaling.Considering the regulatory role of sncRNAs, this newly identified sncRNA signature is likely to reflect the events leading to endometrial cancer, which can be exploited to dissect the carcinogenic process including novel biomarkers for early and non-invasive diagnosis of these tumors.

Identification of H ferritin-dependent and independent genes in K562 differentiating cells by targeted gene silencing and expression profiling.

Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells. In this study we sought to define the repertoire of genes whose expression might be affected by FHC during the hemin-induced differentiation of the erythromyeloid cell line K562. To this aim, gene expression profiling was performed in four different sets of cells: i) wild type K562; ii) sh-RNA FHC-silenced K562; iii) hemin-treated wild-type K562; and iv) hemin-treated FHC-silenced K562. Statistical analysis of the gene expression data, performed by two-factor ANOVA, identified three distinct classes of transcripts: a) Class 1, including 657 mRNAs whose expression is modified exclusively during hemin-induced differentiation of K562 cells, independently from the FHC relative amounts; b) Class 2, containing a set of 70 mRNAs which are consistently modified by hemin and FHC-silencing; and c) Class 3, including 128 transcripts modified by FHC-silencing but not by hemin. Our data indicate that FHC may function as a modulator of gene expression during erythroid differentiation and add new findings to the knowledge of the complex gene network modulated during erythroid differentiation.

Effects of oestrogen on microRNA expression in hormone-responsive breast cancer cells.

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17ß-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.