Despite clear evidence that exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and that exosomal RNA cargo selection is deregulated in pathological conditions, the ...mechanisms controlling specific RNA sorting into extracellular vesicles are still poorly understood. Here, we identified the RNA binding protein SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein; also known as hnRNP-Q or NSAP1) as a component of the hepatocyte exosomal miRNA sorting machinery. SYNCRIP knockdown impairs sorting of miRNAs in exosomes. Furthermore, SYNCRIP directly binds to specific miRNAs enriched in exosomes sharing a common extra-seed sequence (hEXO motif). The hEXO motif has a role in the regulation of miRNA localization, since embedment of this motif into a poorly exported miRNA enhances its loading into exosomes. This evidence provides insights into the mechanisms of miRNA exosomal sorting process. Moreover, these findings open the way for the possible selective modification of the miRNAs exosomal cargo.
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•SYNCRIP interacts specifically with selected miRNAs•SYNCRIP has a functional role in exosomal sorting of specific miRNAs•A hEXO miRNA extra-seed motif determines exosomal loading
Santangelo et al. shed light on the mechanism that allows a cell to load exosomes with a specific repertoire of miRNAs, showing a functional role of a specific miRNA motif and a protein that specifically interacts with it, the RNA-binding protein SYNCRIP.
Background
Several factors may contribute to the circadian variability of clinical manifestations in asthma and allergy. Basophils play a pivotal role in allergic inflammation. However, evidence for ...a functional clock governing the effector function of these cells is sparse and contradictory. We have systematically sampled the 24‐hour response of basophils to IgE‐ and non‐IgE‐dependent ligands in asthma to understand their possible contribution to the diurnal variations of allergic symptoms.
Methods
Leukocytes were collected every 4 hours for 24 hours from 10 patients with moderate, persistent asthma and 10 matched, nonallergic controls, and then incubated with concentrations of anti‐IgE, formyl‐methionyl‐leucylphenylalanine (fMLP), or the Ca2+ ionophore, A23187. Histamine release (HR) was tested for time‐of‐day‐ or disease‐related variability by conventional statistics and for 24‐hour rhythmicity by the cosinor method.
Results
HR induced by anti‐IgE was significantly increased at 08:00 vs. 20:00 in basophils from asthmatics but not controls. No significant differences were seen at any time in the response to A23187, while the response to fMLP was significantly higher at 08:00 vs. 20:00 in controls but not asthmatics. The basophil response to anti‐IgE, but not fMLP or A23187, varied significantly across the 24 hours in asthma, and its amplitude, percent rhythm, and acrophase were comparable to those of peak expiratory flow or serum cortisol.
Conclusion
Using an integrated statistical approach, we show that basophil responsiveness undergoes significant circadian variability and that distinct patterns of rhythmicity can be recognized depending on the signal delivered, the activation parameters assessed, and the disease status.
Basophils were collected serially for 24 hours from asthmatics or controls and activated with IgE‐ or non‐IgE‐mediated secretagogues. Histamine release induced by anti‐IgE exhibited significant circadian rhythmicity in asthma but not controls, in parallel with asynchronous changes in cortisol and PEF levels. Circadian variability of IgE‐dependent basophil responsiveness may contribute to diurnal variability in respiratory function and symptoms in asthma.
Abbreviations: Max % HR, Maximal percent histamine release; PEF, Peak expiratory flow
Abstract
Background
Targeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In ...breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L.
Methods
We combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes.
Results
Dot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine–protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells.
Conclusions
Identification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.
The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, ...transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease.
Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ - cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability.
These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.
The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in ...exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.
Integrins are cell-extracellular matrix adhesion molecules whose expression level undergoes quantitative changes upon neoplastic transformation and are considered functionally related to the ...development of cancer metastasis. We analyzed the largest mRNA-seq dataset available to determine the expression pattern of integrin family subunits in papillary thyroid carcinomas (PTC). ITGA2, 3, 6, V, and ITGB1 integrin subunits were overexpressed in PTC compared to normal thyroid tissue. The PTC histology variants “classical” and “tall cell” displayed a similar integrin expression profile with a higher level of ITGA3, ITGAV, and ITGB1, which differed from that of the “follicular” variant. Interestingly, compared to RAS mutations, BRAFV600E mutation was associated with a significantly higher expression of integrins. Some integrin subunits were associated with advanced disease stage, lymph node metastasis, extrathyroidal extension, and high-risk groups. Among them, ITGA3 expression displayed the highest correlation with advanced disease and was associated with a negative prognosis. In vitro scratch assay and Matrigel invasion assay in two different PTC cell lines confirmed α3β1 role in cell motility and invasion, supporting its involvement during tumor progression. These results demonstrate the existence of a PTC-specific integrin expression signature correlated to histopathology, specific driver gene mutations, and aggressiveness of the disease.
Abstract
Background
Next-Generation-Sequencing (NGS) enables detection of microorganisms present in biological and other matrices of various origin and nature, allowing not only the identification of ...known phyla and strains but also the discovery of novel ones. The large amount of metagenomic shotgun data produced by NGS require comprehensive and user-friendly pipelines for data analysis, that speed up the bioinformatics steps, relieving the users from the need to manually perform complex and time-consuming tasks.
Results
We describe here HOME-BIO (sHOtgun MEtagenomic analysis of BIOlogical entities), an exhaustive pipeline for metagenomics data analysis, comprising three independent analytical modules designed for an inclusive analysis of large NGS datasets.
Conclusions
HOME-BIO is a powerful and easy-to-use tool that can be run also by users with limited computational expertise. It allows in-depth analyses by removing low-complexity/ problematic reads, integrating the analytical steps that lead to a comprehensive taxonomy profile of each sample by querying different source databases, and it is customizable according to specific users’ needs.
The histone lysine methyltransferase DOT1L (DOT1-like histone lysine methyltransferase) is responsible for the epigenetic regulation of gene expression through specific methylation of lysine79 ...residue of histone H3 (H3K79) in actively transcribed genes. Its normal activity is crucial for embryonic development and adult tissues functions, whereas its aberrant functioning is known to contribute to leukemogenesis. DOT1L is the only lysine methyltransferase that does not contain a SET domain, which is a feature that allowed the development of selective DOT1L inhibitors that are currently investigated in Phase I clinical trials for cancer treatment. Recently, abnormal expression of this enzyme has been associated with poor survival and increased aggressiveness of several solid tumors. In this review evidences of aberrant DOT1L expression and activity in breast, ovarian, prostate, colon, and other solid tumors, and its relationships with biological and clinical behavior of the disease and response to therapies, are summarized. Current knowledge of the structural basis of DOT1L ability to regulate cell proliferation, invasion, plasticity and stemness, cell cycle progression, cell-to-cell signaling, epithelial-to-mesenchymal transition, and chemoresistance, through cooperation with several molecular partners including noncoding RNAs, is also reviewed. Finally, available options for the treatment of therapeutically challenging solid tumors by targeting DOT1L are discussed.
In the complex and articulated machinery of the human genome, less than 2% of the transcriptome encodes for proteins, while at least 75% is actively transcribed into non-coding RNAs (ncRNAs). Among ...the non-coding transcripts, those ≥200 nucleotides long (lncRNAs) are receiving growing attention for their involvement in human diseases, particularly cancer. Genomic studies have revealed the multiplicity of processes, including neoplastic transformation and tumor progression, in which lncRNAs are involved by regulating gene expression at epigenetic, transcriptional, and post-transcriptional levels by mechanism(s) that still need to be clarified. In breast cancer, several lncRNAs were identified and demonstrated to have either oncogenic or tumor-suppressive roles. The functional understanding of the mechanisms of lncRNA action in this disease could represent a potential for translational applications, as these molecules may serve as novel biomarkers of clinical use and potential therapeutic targets. This review highlights the relationship between lncRNAs and the principal hallmark of the luminal breast cancer phenotype, estrogen receptor α (ERα), providing an overview of new potential ways to inhibit estrogenic signaling via this nuclear receptor toward escaping resistance to endocrine therapy.
Abstract
Background
Colorectal cancer (CRC) is the third most deadly and fourth most diagnosed cancer worldwide. Despite the progress in early diagnosis and advanced therapeutic options, CRC shows a ...poor prognosis with a 5 year survival rate of ~ 45%.
PRDM2/RIZ
, a member of PR/SET domain family (
PRDM
), expresses two main molecular variants, the PR-plus isoform (
RIZ1
) and the PR-minus (
RIZ2
). The imbalance in their expression levels in favor of
RIZ2
is observed in many cancer types. The full length RIZ1 has been extensively investigated in several cancers where it acts as a tumor suppressor, whereas few studies have explored the RIZ2 oncogenic properties.
PRDM2
is often target of frameshift mutations and aberrant DNA methylation in CRC. However, little is known about its role in CRC.
Methods
We combined in-silico investigation of The Cancer Genome Atlas (TCGA) CRC datasets, cellular and molecular assays, transcriptome sequencing and functional annotation analysis to assess the role of RIZ2 in human CRC.
Results
Our in-silico analysis on TCGA datasets confirmed that
PRDM2
gene is frequently mutated and transcriptionally deregulated in CRC and revealed that a
RIZ2
increase is highly correlated with a significant
RIZ1
downregulation. Then, we assayed several CRC cell lines by qRT-PCR analysis for the main
PRDM2
transcripts and selected DLD1 cell line, which showed the lowest
RIZ2
levels. Therefore, we overexpressed
RIZ2
in these cells to mimic TCGA datasets analysis results and consequently to assess the
PRDM2/RIZ2
role in CRC. Data from RNA-seq disclosed that
RIZ2
overexpression induced profound changes in CRC cell transcriptome via EGF pathway deregulation, suggesting that RIZ2 is involved in the EGF autocrine regulation of DLD1 cell behavior. Noteworthy, the forced
RIZ2
expression increased cell viability, growth, colony formation, migration and organoid formation. These effects could be mediated by the release of high EGF levels by
RIZ2
overexpressing DLD1 cells.
Conclusions
Our findings add novel insights on the putative RIZ2 tumor-promoting functions in CRC, although additional efforts are warranted to define the underlying molecular mechanism.
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