Ribonuclease inhibitor and angiogenin system regulates cell type-specific global translation.

Translation of mRNAs is a fundamental process that occurs in all cell types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell type-specific translation. Despite this, it remains unclear whether global translation is controlled in a cell type-specific manner. By using human cell lines and mouse models, we found that deletion of the ribosome-associated protein ribonuclease inhibitor 1 (RNH1) decreases global translation selectively in hematopoietic-origin cells but not in the non-hematopoietic-origin cells. RNH1-mediated cell type-specific translation is mechanistically linked to angiogenin-induced ribosomal biogenesis. Collectively, this study unravels the existence of cell type-specific global translation regulators and highlights the complex translation regulation in vertebrates.

Molecular characterization of breast cancer cell pools with normal or reduced ability to respond to progesterone: a study based on RNA-seq.

BACKGROUND: About one-third of patients with estrogen receptor alpha (ERα)-positive breast cancer have tumors which are progesterone receptor (PR) negative. PR is an important prognostic factor in breast cancer. Patients with ERα-positive/PR-negative tumors have shorter disease-free and overall survival than patients with ERα-positive/PR-positive tumors. New evidence has shown that progesterone (P4) has an anti-proliferative effect in ERα-positive breast cancer cells. However, the role of PR in breast cancer is only poorly understood. METHODS: We disrupted the PR gene (PGR) in ERα-positive/PR-positive T-47D cells using the CRISPR/Cas9 system. This resulted in cell pools we termed PR-low as P4 mediated effects were inhibited or blocked compared to control T-47D cells. We analyzed the gene expression profiles of PR-low and control T-47D cells in the absence of hormone and upon treatment with P4 alone or P4 together with estradiol (E2). Differentially expressed (DE) genes between experimental groups were characterized based on RNA-seq and Gene Ontology (GO) enrichment analyses. RESULTS: The overall gene expression pattern was very similar between untreated PR-low and untreated control T-47D cells. More than 6000 genes were DE in control T-47D cells upon stimulation with P4 or P4 plus E2. When PR-low pools were subjected to the same hormonal treatment, up- or downregulation was either blocked/absent or consistently lower. We identified more than 3000 genes that were DE between hormone-treated PR-low and control T-47D cells. GO analysis revealed seven significantly enriched biological processes affected by PR and associated with G protein-coupled receptor (GPCR) pathways which have been described to support growth, invasiveness, and metastasis in breast cancer cells. CONCLUSIONS: The present study provides new insights into the complex role of PR in ERα-positive/PR-positive breast cancer cells. Many of the genes affected by PR are part of central biological processes of tumorigenesis.

CNS Antigen-Specific Neuroinflammation Attenuates Ischemic Stroke With Involvement of Polarized Myeloid Cells.

BACKGROUND AND OBJECTIVES: Experimental studies indicate shared molecular pathomechanisms in cerebral hypoxia-ischemia and autoimmune neuroinflammation. This has led to clinical studies investigating the effects of immunomodulatory therapies approved in multiple sclerosis on inflammatory damage in stroke. So far, mutual and combined interactions of autoimmune, CNS antigen-specific inflammatory reactions and cerebral ischemia have not been investigated so far. METHODS: Active MOG35-55 experimental autoimmune encephalomyelitis (EAE) was induced in male C57Bl/6J mice. During different phases of EAE, transient middle cerebral artery occlusion (tMCAO, 60 minutes) was induced. Brain tissue was analyzed for infarct size and immune cell infiltration. Multiplex gene expression analysis was performed for 186 genes associated with neuroinflammation and hypoxic-ischemic damage. RESULTS: Mice with severe EAE disease showed a substantial reduction in infarct size after tMCAO. Histopathologic analysis showed less infiltration of CD45+ hematopoietic cells in the infarct core of severely diseased acute EAE mice; this was accompanied by an accumulation of Arginase1-positive/Iba1-positive cells. Gene expression analysis indicated an involvement of myeloid cell-driven anti-inflammatory mechanisms in the attenuation of ischemic injury in severely diseased mice exposed to tMCAO in the acute EAE phase. DISCUSSION: CNS autoantigen-specific autoimmunity has a protective influence on primary tissue damage after experimental stroke, indicating a very early involvement of CNS antigen-specific, myeloid cell-associated anti-inflammatory immune mechanisms that mitigate ischemic injury in the acute EAE phase.

Impact of 6 month conjugated equine estrogen versus estradiol-treatment on biomarkers and enriched gene sets in healthy mammary tissue of non-human primates.

OBJECTIVE: To identify distinctly regulated gene markers and enriched gene sets in breast tissue of cynomolgus monkeys (Macaca fascicularis) treated for six months with either conjugated equine estrogens (CEE) or estradiol (E2) by analysis of corresponding mRNA levels of genes associated with breast development, carcinogenesis, apoptosis and immune regulation. Additionally, translation of three nuclear markers was analyzed. METHODS: RNA from breast biopsies and necropsies was isolated from two independent study trials from Ethun et al. (CEE) and Foth et al. (E2) after 6 month of treatment duration. RNA was subjected to qRT-PCR and MicroArray analysis. Immunohistochemical stainings were performed for the estrogen receptor alpha subunit (ERa), the progesterone receptor (PGR) and the proliferation marker Ki67. RESULTS: We identified a total of 36 distinctly enriched gene sets. Thirty-one were found in the CEE treatment group and five were found in the E2 treatment group, with no overlap. Furthermore, two individual genes IGFBP1 and SGK493 were upregulated in CEE treated animals. Additional targeted qRT-PCR analysis of ten specific estrogen-related genes showed upregulation of three genes (TFF1, PGR and GREB1) after CEE treatment, respectively one gene (TFF1) after E2 treatment. Immunohistochemical stains of breast biopsies showed a significant increase in expression of the PGR marker after CEE treatment. CONCLUSIONS: In this study we identified enriched gene sets possibly induced by CEE or E2 treatment in various processes associated with cancer biology and immunology. This preliminary translational data supports the concept that different estrogen types have different effects on healthy breast tissue and may help generate hypotheses for future research.

Integrated mRNA-miRNA transcriptome analysis of bladder biopsies from patients with bladder pain syndrome identifies signaling alterations contributing to the disease pathogenesis.

BACKGROUND: Interstitial cystitis, or bladder pain syndrome (IC/BPS), is a chronic bladder disorder characterized by lower abdominal pain associated with the urinary bladder and accompanied by urinary frequency and urgency in the absence of identifiable causes. IC/PBS can be separated into the classic Hunner's ulcerative type and the more prevalent non-ulcerative disease. Our aim was to unravel the biological processes and dysregulated cell signaling pathways leading to the bladder remodeling in non-ulcerative bladder pain syndrome (BPS) by studying the gene expression changes in the patients' biopsies. METHODS: We performed paired microRNA (miRNA) and mRNA expression profiling in the bladder biopsies of BPS patients with non-Hunner interstitial cystitis phenotype, using comprehensive Next-generation sequencing (NGS) and studied the activated pathways and altered biological processes based on the global gene expression changes. Paired mRNA-miRNA transcriptome analysis delineated the regulatory role of the dysregulated miRNAs by identifying their targets in the disease-induced pathways. RESULTS: EIF2 Signaling and Regulation of eIF4 and p70S6K Signaling, activated in response to cellular stress, were among the most significantly regulated processes during BPS. Leukotriene Biosynthesis nociceptive pathway, important in inflammatory diseases and neuropathic pain, was also significantly activated. The biological processes identified using Gene Ontology over-representation analysis were clustered into six main functional groups: cell cycle regulation, chemotaxis of immune cells, muscle development, muscle contraction, remodeling of extracellular matrix and peripheral nervous system organization and development. Compared to the Hunner's ulcerative type IC, activation of the immune pathways was modest in non-ulcerative BPS, limited to neutrophil chemotaxis and IFN-γ-mediated signaling. We identified 62 miRNAs, regulated and abundant in BPS and show that they target the mRNAs implicated in eIF2 signalling pathway. CONCLUSIONS: The bladders of non-ulcerative BPS patients recruited in this study had alterations consistent with a strong cell proliferative response and an up-regulation of smooth muscle contractility, while the contribution of inflammatory processes was modest. Pathway analysis of the integrated mRNA-miRNA NGS dataset pinpointed important regulatory miRNAs whose dysregulation might contribute to the pathogenesis. Observed molecular changes in the peripheral nervous system organization and development indicate the potential role of local bladder innervation in the pain perceived in this type of BPS.

Understanding the Interactions Between the Ocular Surface Microbiome and the Tear Proteome.

Purpose: The purpose of this study was to explore the interplay between the ocular surface microbiome and the tear proteome in humans in order to better understand the pathogenesis of ocular surface-associated diseases. Methods: Twenty eyes from 20 participants were included in the study. The ocular surface microbiome was sequenced by whole-metagenome shotgun sequencing using lid and conjunctival swabs. Furthermore, the tear proteome was identified using chromatography tandem mass spectrometry. After compositional and functional profiling of the metagenome and functional characterization of the proteome by gene ontology, association studies between the ocular microbiome and tear proteome were assessed. Results: Two hundred twenty-nine taxa were identified with Actinobacteria and Proteobacteria being the most abundant phyla with significantly more Propionibacterium acnes and Staphylococcus epidermidis in lid compared to conjunctival swabs. The lid metagenomes were enriched in genes of the glycolysis lll and adenosine nucleotides de novo and L-isoleucine biosynthesis. Correlations between the phylum Firmicutes and fatty acid metabolism, between the genus Agrobacterium as well as vitamin B1 synthesis and antimicrobial activity, and between biosynthesis of heme, L-arginine, as well as L-citrulline and human vision were detected. Conclusions: The ocular surface microbiome was found to be associated with the tear proteome with a role in human immune defense. This study has a potential impact on the development of treatment strategies for ocular surface-associated diseases.

TIRAP p.R81C is a novel lymphoma risk variant which enhances cell proliferation via NF-κB mediated signaling in B-cells.

Diffuse large B-cell lymphoma is the most common malignant lymphoma in adults. By gene-expression profiling, this lymphoma is divided in three cell-of-origin subtypes with distinct molecular and clinical features. Most lymphomas arise sporadically, yet familial clustering is known, suggesting a genetic contribution to disease risk. Familial lymphoma cases are a valuable tool to investigate risk genes. We studied a Swiss/Japanese family with 2 sisters affected by a primary mediastinal B-cell lymphoma and a non-germinal center diffuse large B-cell lymphoma not otherwise specified, respectively. The somatic landscape of both lymphomas was marked by alterations affecting multiple components of the JAK-STAT pathway. Consequently, this pathway was constitutively activated as evidenced by high pJAK2 as well as increased nuclear pSTAT3 and pSTAT6 in malignant cells. Potential lymphoma risk variants were identified by whole exome sequencing of the germline DNA derived from siblings and unaffected family members. This analysis revealed a pathogenic variant in TIRAP, an upstream regulator of NF-κB, in both affected siblings and their mother. We observed increased B-cell proliferation in family members harboring the TIRAP p.R81C variant. B-cell proliferation correlated with TIRAP and NF-κB target gene expression, suggesting enhanced NF-κB pathway activity in TIRAP p.R81C individuals. TIRAP knockdown reduced B-cell survival and NF-κB target gene expression, particularly in individuals with TIRAP p.R81C. Functional studies revealed significantly increased NF-κB activity and resistance to stress-induced cell-death by TIRAP p.R81C. The identification of an inherited TIRAP variant provides evidence for a novel link between genetic alterations affecting the NF-κB pathway and lymphomagenesis.

Gene expression in chronic granulomatous disease and interferon-γ receptor-deficient cells treated in vitro with interferon-γ.

Interferon-γ (IFN-γ) plays an important role in innate and adaptive immunity against intracellular infections and is used clinically for the prevention and control of infections in chronic granulomatous disease (CGD) and inborn defects in the IFN-γ/interleukin (IL)-12 axis. Using transcriptome profiling (RNA-seq), we sought to identify differentially expressed genes, transcripts and exons in Epstein-Barr virus-transformed B lymphocytes (B-EBV) cells from CGD patients, IFN-γ receptor deficiency patients, and normal controls, treated in vitro with IFN-γ for 48 hours. Our results show that IFN-γ increased the expression of a diverse array of genes related to different cellular programs. In cells from normal controls and CGD patients, IFN-γ-induced expression of genes relevant to oxidative killing, nitric oxide synthase pathway, proteasome-mediated degradation, antigen presentation, chemoattraction, and cell adhesion. IFN-γ also upregulated genes involved in diverse stages of messenger RNA (mRNA) processing including pre-mRNA splicing, as well as others implicated in the folding, transport, and assembly of proteins. In particular, differential exon expression of WARS (encoding tryptophanyl-transfer RNA synthetase, which has an essential function in protein synthesis) induced by IFN-γ in normal and CGD cells suggests that this gene may have an important contribution to the benefits of IFN-γ treatment for CGD. Upregulation of mRNA and protein processing related genes in CGD and IFNRD cells could mediate some of the effects of IFN-γ treatment. These data support the concept that IFN-γ treatment may contribute to increased immune responses against pathogens through regulation of genes important for mRNA and protein processing.

Osteolytic cancer cells induce vascular/axon guidance processes in the bone/bone marrow stroma.

Prostate and breast cancers frequently metastasize to bone. The physiological bone homeostasis is perturbed once cancer cells proliferate at the bone metastatic site. Tumors are complex structures consisting of cancer cells and numerous stroma cells. In this study, we show that osteolytic cancer cells (PC-3 and MDA-MB231) induce transcriptome changes in the bone/bone marrow microenvironment (stroma). This stroma transcriptome differs from the previously reported stroma transcriptome of osteoinductive cancer cells (VCaP). While the biological process "angiogenesis/vasculogenesis" is enriched in both transcriptomes, the "vascular/axon guidance" process is a unique process that characterizes the osteolytic stroma. In osteolytic bone metastasis, angiogenesis is denoted by vessel morphology and marker expression specific for arteries/arterioles. Interestingly, intra-tumoral neurite-like structures were in proximity to arteries. Additionally, we found that increased numbers of mesenchymal stem cells and vascular smooth muscle cells, expressing osteolytic cytokines and inhibitors of bone formation, contribute to the osteolytic bone phenotype. Osteoinductive and osteolytic cancer cells induce different types of vessels, representing functionally different hematopoietic stem cell niches. This finding suggests different growth requirements of osteolytic and osteoinductive cancer cells and the need for a differential anti-angiogenic strategy to inhibit tumor growth in osteolytic and osteoblastic bone metastasis.

The ESRP1-GPR137 axis contributes to intestinal pathogenesis.

Aberrant alternative pre-mRNA splicing (AS) events have been associated with several disorders. However, it is unclear whether deregulated AS directly contributes to disease. Here, we reveal a critical role of the AS regulator epithelial splicing regulator protein 1 (ESRP1) for intestinal homeostasis and pathogenesis. In mice, reduced ESRP1 function leads to impaired intestinal barrier integrity, increased susceptibility to colitis and altered colorectal cancer (CRC) development. Mechanistically, these defects are produced in part by modified expression of ESRP1-specific Gpr137 isoforms differently activating the Wnt pathway. In humans, ESRP1 is downregulated in inflamed biopsies from inflammatory bowel disease patients. ESRP1 loss is an adverse prognostic factor in CRC. Furthermore, generation of ESRP1-dependent GPR137 isoforms is altered in CRC and expression of a specific GPR137 isoform predicts CRC patient survival. These findings indicate a central role of ESRP1-regulated AS for intestinal barrier integrity. Alterations in ESRP1 function or expression contribute to intestinal pathology.

The hyaluronan-mediated motility receptor RHAMM promotes growth, invasiveness and dissemination of colorectal cancer.

In colorectal cancer (CRC), RHAMM is an independent adverse prognostic factor. The aim of the study was therefore to investigate on the role of RHAMM as a potential direct driver of cell proliferation and migration in CRC cell lines and to identify pathways dependent on RHAMM in human CRC. Proliferation, cell cycle alterations and invasive capacity were tested in two RHAMM- and control- knockdown CRC cell lines by flow cytometry and in vitro assays. Tumorigenicity and metastasis formation was assessed in immunodeficient mice. RNA-Seq and immunohistochemistry was performed on six RHAMM+/- primary CRC tumors. In vitro, silencing of RHAMM inhibited CRC cell migration and invasion by 50% (p<0.01). In vivo, RHAMM knockdown resulted in slower growth, lower tumor size (p<0.001) and inhibition of metastasis (p<0.001). Patients with RHAMM-high CRC had a worse prognosis (p=0.040) and upregulated pathways for cell cycle progression and adhesion turnover. RHAMM overexpression is correlated with increased migration and invasion of CRC cells, leads to larger, fast growing tumors, and its downregulation essentially abolishes metastasis in mouse models. RHAMM is therefore a promising therapeutic target in all CRC stages as its inhibition affects growth and dissemination of the primary CRC as well as the metastases.

Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction.

Bladder outlet obstruction (BOO) induces significant organ remodeling, leading to lower urinary tract symptoms accompanied by urodynamic changes in bladder function. Here, we report mRNA and miRNA transcriptome sequencing of bladder samples from human patients with different urodynamically defined states of BOO. Patients' miRNA and mRNA expression profiles correlated with urodynamic findings. Validation of RNA sequencing results in an independent patient cohort identified combinations of 3 mRNAs (NRXN3, BMP7, UPK1A) and 3 miRNAs (miR-103a-3p, miR-10a-5p, miR-199a-3p) sufficient to discriminate between bladder functional states. All BOO patients shared cytokine and immune response pathways, TGF-ß and NO signaling pathways, and hypertrophic PI3K/AKT signaling pathways. AP-1 and NFkB were dominant transcription factors, and TNF-α was the top upstream regulator. Integrated miRNA-mRNA expression analysis identified pathways and molecules targeted by differentially expressed miRNAs. Molecular changes in BOO suggest an increasing involvement of miRNAs in the control of bladder function from the overactive to underactive/acontractile states.

Characterization and Transcriptomic Analysis of Porcine Blood Conventional and Plasmacytoid Dendritic Cells Reveals Striking Species-Specific Differences.

Porcine dendritic cells (DCs) are relatively well characterized, but a clear-cut identification of all DC subsets combined with full transcriptional profiling was lacking, preventing an unbiased insight into the functional specializations of DC subsets. Using a large panel of Abs in multicolor flow cytometry, cell sorting, and RNA sequencing we identified and characterized the porcine equivalent of conventional DCs (cDC) 1 and cDC2 as well as plasmacytoid DCs (pDCs) in the peripheral blood of pigs. We demonstrate that cDC1 are CD135+CD14-CD172alowCADM1+wCD11R1+ cells, cDC2 are CD135+CD14-CD172a+CADM1+CD115+wCD11R1+CD1+ cells and pDCs are CD4+CD135+CD172a+CD123+CD303+ cells. As described in other species, only cDC1 express BATF3 and XCR1, cDC2 express FCER1A and FCGR2B, and only pDCs express TCF4 and NRP1 Nevertheless, despite these cross-species conserved subset-specific transcripts, porcine pDCs differed from the species described so far in many expressed genes and transcriptomic profiling clustered pDCs more distantly from cDCs than monocytes. The response of porcine DC subsets to TLR ligands revealed that pDCs are by far the most important source of TNF-α, IL-12p40, and of course IFN-α, whereas cDCs are most efficient in MHC and costimulatory molecule expression. Nevertheless, upregulation of CD40 and CD86 in cDCs was critically influenced or even dependent on the presence of pDCs, particularly for TLR 7 and 9 ligands. Our data demonstrate that extrapolation of data on DC biology from one species to another has to be done with care, and it shows how functional details have evolved differentially in different species.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.

BACKGROUND & AIMS: Unhealthy lifestyles predispose people to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. METHODS: Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 min/day, 5 days/week during 32 weeks. RESULTS: After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3)vs. 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. CONCLUSIONS: These data show a beneficial effect of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.

The genomic substrate for adaptive radiation in African cichlid fish.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Genome-wide RAD sequence data provide unprecedented resolution of species boundaries and relationships in the Lake Victoria cichlid adaptive radiation.

Although population genomic studies using next generation sequencing (NGS) data are becoming increasingly common, studies focusing on phylogenetic inference using these data are in their infancy. Here, we use NGS data generated from reduced representation genomic libraries of restriction-site-associated DNA (RAD) markers to infer phylogenetic relationships among 16 species of cichlid fishes from a single rocky island community within Lake Victoria's cichlid adaptive radiation. Previous attempts at sequence-based phylogenetic analyses in Victoria cichlids have shown extensive sharing of genetic variation among species and no resolution of species or higher-level relationships. These patterns have generally been attributed to the very recent origin (<15,000 years) of the radiation, and ongoing hybridization between species. We show that as we increase the amount of sequence data used in phylogenetic analyses, we produce phylogenetic trees with unprecedented resolution for this group. In trees derived from our largest data supermatrices (3 to >5.8 million base pairs in width), species are reciprocally monophyletic with high bootstrap support, and the majority of internal branches on the tree have high support. Given the difficulty of the phylogenetic problem that the Lake Victoria cichlid adaptive radiation represents, these results are striking. The strict interpretation of the topologies we present here warrants caution because many questions remain about phylogenetic inference with very large genomic data set and because we can with the current analysis not distinguish between effects of shared ancestry and post-speciation gene flow. However, these results provide the first conclusive evidence for the monophyly of species in the Lake Victoria cichlid radiation and demonstrate the power that NGS data sets hold to resolve even the most difficult of phylogenetic challenges.