[Show abstract][Hide abstract] ABSTRACT: In-vitro expansion of functional adult human β-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of β-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded β-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), β-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human β-like cells for cell therapy of diabetes.
[Show abstract][Hide abstract] ABSTRACT: Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina.
[Show abstract][Hide abstract] ABSTRACT: Molecular mechanisms that regulate lung repair vs. progressive scarring in pulmonary fibrosis remain elusive. Interleukin (IL)-4 and IL-13 are pro-fibrotic cytokines that share common receptor chains including IL-13 receptor (R) α1 and are key pharmacological targets in fibrotic diseases. However, the roles of IL-13Rα1 in mediating lung injury/repair are unclear. We report dysregulated levels of IL-13 receptors in the lungs of bleomycin-treated mice and to some extent in idiopathic pulmonary fibrosis patients. Transcriptional profiling demonstrated an epithelial cell-associated gene signature that was homeostatically dependent on IL-13Rα1 expression. IL-13Rα1 regulated a striking array of genes in the lung following bleomycin administration and Il13ra1 deficiency resulted in exacerbated bleomycin-induced disease. Increased pathology in bleomycin-treated Il13ra1(-/-) mice was due to IL-13Rα1 expression in structural and hematopoietic cells but not due to increased responsiveness to IL-17, IL-4, IL-13, increased IL-13Rα2 or type 1 IL-4R signaling. These data highlight underappreciated protective roles for IL-13Rα1 in lung injury and homeostasis.Mucosal Immunology advance online publication, 8 July 2015; doi:10.1038/mi.2015.56.
[Show abstract][Hide abstract] ABSTRACT: Alopecia-Neurological defects-Endocrinopathy (ANE) syndrome is a rare inherited hair disorder, which was shown to result from decreased expression of the RNA binding motif protein 28 (RBM28). In the present study, we attempted to delineate the role of RBM28 in hair biology. First, we sought to obtain evidence for the direct involvement of RBM28 in hair growth. When RBM28 was down-regulated in human hair follicle (HF) organ cultures, we observed catagen induction and HF growth arrest, indicating that RBM28 is necessary for normal hair growth. We also aimed at identifying molecular targets of RBM28. Given that an RBM28 homolog was recently found to regulate miRNA biogenesis in C. elegans and given the known pivotal importance of miRNAs for proper hair follicle development, we studied global miRNA expression profile in cells knocked down for RBM28. This analysis revealed that RBM28 controls the expression of miR-203. miR-203 was found to regulate in turn TP63, encoding the transcription factor p63, which is critical for hair morphogenesis. In conclusion, RBM28 contributes to HF growth regulation through modulation of miR-203 and p63 activity. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Genetic syndromes involving both brain and eye abnormalities are numerous and include syndromes such as Warburg micro syndrome, Kaufman oculocerebrofacial syndrome, Cerebro-oculo-facio-skeletal syndrome, Kahrizi syndrome and others. Using exome sequencing, we have been able to identify homozygous mutation p.(Tyr39Cys) in MED25 as the cause of a syndrome characterized by eye, brain, cardiac and palatal abnormalities as well as growth retardation, microcephaly and severe intellectual disability in seven patients from four unrelated families, all originating from the same village. The protein encoded by MED25 belongs to Mediator complex or MED complex, which is an evolutionary conserved multi-subunit RNA polymerase II transcriptional regulator complex. The MED25 point mutation is located in the von Willebrand factor type A (MED25 VWA) domain which is responsible for MED25 recruitment into the Mediator complex; co-immunoprecipitation experiment demonstrated that this mutation dramatically impairs MED25 interaction with the Mediator complex in mammalian cells.
Human Genetics 03/2015; 134(6). DOI:10.1007/s00439-015-1541-x · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Placental factors, progesterone included, facilitate breast cancer cell line (BCCL) motility and thus may contribute to the advanced breast cancer found during pregnancy. Cancer and placental implantations are similar; the last is accompanied by extravillous trophoblast cell invasion and autophagy which are interlinked. We aimed to analyze the effect of first trimester human placenta on BCCL autophagy. BCCLs (MCF-7/T47D) were cultured with placental explants (60 h) or placental supernatants (24 h). Following cultures, BCCLs were sorted out for RNA/protein extraction. RNA served for microarray/qPCR (BNIP3) and protein for Western blot (HIF1α, LC3BII) analyses. Inhibitors were added to the placenta-MCF-7 coculture or placental supernatants (autophagy inhibitor-3MA, progesterone receptor (PR) inhibitor-RU486, and HIF1α inhibitor-Vitexin) in order to evaluate their effects on BCCL motility and LC3BII/HIF1α expression. LC3BII (an autophagy marker) expression was elevated in BCCLs following placental explant coculture and exposure to placental supernatants. The autophagy inhibitor (3MA) repressed the placenta-induced MCF-7/T47D migration, establishing a connection between BCCL autophagy and migration. Microarray analysis of MCF-7 following placenta-MCF-7 coculture showed that "HIF1α pathway," a known autophagy facilitator, was significantly manipulated. Indeed, placental factors elevated HIF1α and its target BNIP3 in the BCCLs, verifying array results. Lastly, PR inhibitor reduced HIF1α expression and both PR and HIF1α inhibitors reduced MCF-7 LC3BII expression and motility, suggesting involvement of the PR-HIF1α axis in the autophagy process. Placental factors induced BCCL autophagy that is interlinked to their motility. This suggests that autophagy-related molecules may serve as targets for therapy in pregnancy-associated breast cancer.
Breast Cancer Research and Treatment 02/2015; 149(3). DOI:10.1007/s10549-015-3266-x · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accumulating data indicate translation plays a role in cancer biology, particularly its rate limiting stage of initiation. Despite this evolving recognition, the function and importance of specific translation initiation factors is unresolved. The eukaryotic translation initiation complex eIF4F consists of eIF4E and eIF4G at a 1:1 ratio. Although it is expected that they display interdependent functions, several publications suggest independent mechanisms. This study is the first to directly assess the relative contribution of eIF4F components to the expressed cellular proteome, transcription factors, microRNAs, and phenotype in a malignancy known for extensive protein synthesis-multiple myeloma (MM). Previously, we have shown that eIF4E/eIF4GI attenuation (siRNA/Avastin) deleteriously affected MM cells' fate and reduced levels of eIF4E/eIF4GI established targets. Here, we demonstrated that eIF4E/eIF4GI indeed have individual influences on cell proteome. We used an objective, high throughput assay of mRNA microarrays to examine the significance of eIF4E/eIF4GI silencing to several cellular facets such as transcription factors, microRNAs and phenotype. We showed different imprints for eIF4E and eIF4GI in all assayed aspects. These results promote our understanding of the relative contribution and importance of eIF4E and eIF4GI to the malignant phenotype and shed light on their function in eIF4F translation initiation complex.
[Show abstract][Hide abstract] ABSTRACT: The recent finding of activity-dependent neuroprotective protein (ADNP) as a protein decreased in serum of patients with Alzheimer's disease (AD) compared to controls, alongside with the discovery of ADNP mutations in autism and coupled with the original description of cancer mutations, ignited an interest for a comparative analysis of ADNP with other AD/autism/cancer-associated genes. We strive toward a better understanding of the molecular structure of key players in psychiatric/neurodegenerative diseases including autism, schizophrenia, and AD. This article includes data mining and bioinformatics analysis on the ADNP gene and protein, in addition to other related genes, with emphasis on recent literature. ADNP is discovered here as unique to chordata with specific autism mutations different from cancer-associated mutation. Furthermore, ADNP exhibits similarities to other cancer/autism-associated genes. We suggest that key genes, which shape and maintain our brain and are prone to mutations and are by in large unique to chordata. Furthermore, these brain-controlling genes, like ADNP, are linked to cell growth and differentiation, and under different stress conditions may mutate or exhibit expression changes leading to cancer propagation. Better understanding of these genes could lead to better therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Abstract Background: Macrophages are heterogeneous cells, which possess pleotropic effector and immunoregulatory functions. The phenotypic diversity of macrophages is best exemplified by the ability of IL-4 or IL-13, two key cytokines in asthma to promote macrophages into a suppressive/anti-inflammatory phenotype (e.g. alternatively activated or M2) whereas exposure to IFN-γ followed by microbial trigger renders macrophages pro-inflammatory (e.g. classically activated or M1). Intriguingly, only limited data exists regarding the expression of miRNA in M2 macrophages. Objective: To define the miRNA profile of M2 and M1 macrophages. Methods: Bone marrow-derived macrophages were activated to classically and alternatively activated states using IL-4, IL-13 or IFN-γ followed by E. Coli stimulation. Thereafter, an unbiased miRNA "mining" approach was utilized and the expression of several miRNAs was validated following in-vitro and in-vivo macrophage activation (qPCR). miR-511 over-expression was performed followed by global transcriptional and bioinformatic analyses. Results: We report unique miRNA expression profiles in M2 and M1 macrophages involving multiple miRNAs. Among these miRNAs we establish that miR-511 is increased in macrophages following IL-4- and IL-13-stimulation and decreased in M1 macrophages both in-vitro and in-vivo. Increased miR-511 expression was sufficient to induce marked transcriptional changes in macrophages. Interestingly, bioinformatics analyses revealed that miR-511 altered the expression of gene products that are associated with hallmark alternatively activated macrophage functions such as cellular proliferation, wound healing responses and inflammation. Conclusions: Our data establish miR-511 as a bona fide M2-associated miRNA. These data may have significant implications in asthma where the expression of IL-4 and IL-13 are highly increased.
Journal of Asthma 11/2014; 52(6):1-25. DOI:10.3109/02770903.2014.988222 · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Women diagnosed with pregnancy associated breast cancer often have advanced cancer with metastases and reduced expression of ERα compared to non-pregnant women. Nevertheless, metastases to the placenta are uncommon. Previously, we demonstrated that breast cancer cells (MCF-7/T47D) migrated from ex vivo human placental explant implantation sites. We aimed to analyze the effect of factors produced during placental implantation or as a result of the interaction between the implanted placentae to cancer cells on cancer cells migration and aggressiveness. We collected supernatants from implanted placentae and placental-breast cancer cells cocultures and analyzed their effects on cancer cells phenotype and pathways. Supernatants collected from breast cancer cells served as controls. We found that supernatants collected from implanted placentae induced modest cancer cells migration that was not accompanied by epithelial to mesenchymal transition (EMT), supported breast cancer cells survival and elevated MCF-7 cell number. The coculture supernatant induced excessive motility and EMT of the MCF-7 cells. This EMT was mediated by Smad3 and JNK/ERK activation. Both placenta and coculture supernatants reduced ERα expression in the cancer cells. Finally, we showed that MCF-7 cocultured with the human placental explants underwent continuous activation of JNK and Smad3 pathways and the EMT process, which led to their migration away from the placental implantation sites. These findings may explain the reduced ERα and elevated metastases found in breast cancer during pregnancy and highlights pathways involved in it.
[Show abstract][Hide abstract] ABSTRACT: Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cell carrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL fusion gene. Tyrosine kinase inhibitors (TKIs) of the BCR-ABL kinase are the treatment of choice for CML patients. Imatinib was the first TKI used in clinical practice with excellent results. MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. Aberrant miRNA expression profiles have been shown to be characteristic of many cancers. Here, we demonstrate that miR-30e is expressed at low levels in CML cell lines and patient samples. Bioinformatics analysis reveals a putative target site for miR-30e in the 3'-untranslated region (UTR) of the ABL gene. In agreement, luciferase assay verified that miR-30e directly targets ABL. Enforced expression of miR-30e in K562 cells suppressed proliferation and induced apoptosis of these cells and sensitized them to imatinib treatment. These findings strongly suggest that miR-30e acts as a tumor suppressor by down regulating BCR-ABL expression. Up-regulation of miR-30e in CML cells may therefore have a therapeutic efficacy against this disease.
Cancer Letters 10/2014; 356(2). DOI:10.1016/j.canlet.2014.10.006 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives:
To evaluate molecular profiles in the small bowel (SB) mucosa proximal to the pouch in ulcerative colitis (UC) patients after pouch surgery.
Patients were prospectively recruited and stratified according to disease behaviour: normal pouch (NP), chronic pouchitis (CP), and Crohn's-like disease of the pouch (CLDP). Biopsies obtained from the pouch and the normal-appearing proximal SB (40 cm proximal to the anal verge) were compared to ileal biopsies from normal controls (NC). A histopathological score based on the degree of polymorphonuclear and mononuclear infiltrates was used to assess inflammation in the pouch and the proximal SB. Gene expression analysis was performed using microarrays, and validated by real-time PCR. Gene ontology and clustering were evaluated by bioinformatics.
Thirty-six subjects were recruited (age 18-71 years, 16 males). Histopathology scores demonstrated minimal differences in the normal-appearing proximal SB of all groups. Nonetheless, significant (fold change ≥2, corrected p [FDR] ≤ 0.05) molecular alterations in the proximal SB were detected in all groups (NP n=9; CP n=80; and CLDP n=230) compared with NC. The magnitude of DUOX2 alteration in the proximal SB was highest. An increase of 6.0, 9.8 and 21.7 folds in DUOX2 expression in NP, CP, CLDP, respectively was observed. This was followed by alterations in MMP1, SLC6A14 and PGC. Gene alterations in the proximal SB overlapped with alterations within the pouch (76% and 97% overlap in CP and CLDP, respectively). Gene ontology analysis in the proximal SB and pouch were comparable.
Significant gene expression alterations exist in an apparently unaffected proximal SB. Alterations in the pouch and the proximal SB were comparable, suggesting that inflammation may not be limited to the pouch, but that it extends to the proximal SB.
Gut 06/2014; 64(5). DOI:10.1136/gutjnl-2014-307387 · 14.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The liver has a remarkable capacity to regenerate after injury; yet, the role of macrophages (MF) in this process remains controversial mainly due to difficulties in distinguishing between different MF subsets. In this study, we used a murine model of acute liver injury induced by overdose of N-acetyl-p-aminophenol (APAP) and defined three distinct MF subsets that populate the liver following injury. Accordingly, resident Kupffer cells (KC) were significantly reduced upon APAP challenge and started recovering by self-renewal at resolution phase without contribution of circulating Ly6C(hi) monocytes. The latter were recruited in a CCR2- and M-CSF-mediated pathway at the necroinflammatory phase and differentiated into ephemeral Ly6C(lo) MF subset at resolution phase. Moreover, their inducible ablation resulted in impaired recovery. Microarray-based molecular profiling uncovered high similarity between steady-state KC and those recovered at the resolution phase. In contrast, KC and monocyte-derived MF displayed distinct prorestorative genetic signature at the resolution phase. Finally, we show that infiltrating monocytes acquire a prorestorative polarization manifested by unique expression of proangiogenesis mediators and genes involved with inhibition of neutrophil activity and recruitment and promotion of their clearance. Collectively, our results present a novel phenotypic, ontogenic, and molecular definition of liver-MF compartment following acute injury.
The Journal of Immunology 06/2014; 193(1). DOI:10.4049/jimmunol.1400574 · 4.92 Impact Factor