MicroRNAs in CD4+ T cell subsets are markers of disease risk and T cell dysfunction in individuals at risk for type 1 diabetes

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4⁺ FOXP3⁺ T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4⁺ T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4⁺ T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4⁺ T cell subsets are markers of risk and T cell dysfunction in T1D.     

Gene and miRNA Expression Profiles of Hematopoietic Progenitor Cells Vary Depending on Their Origin

Hematopoietic progenitor cells (HPCs) from granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood (G-PB), bone marrow (BM), or umbilical cord blood (CB) have differing biological properties and differing kinetics of engraftment post-transplantation, which might be explained, at least in part, by differing gene and miRNA expression patterns. To assess the differences in gene and miRNA expression, we analyzed whole genome expression profiles as well as the expression of 384 miRNAs in CD34⁺ cells isolated from 18 healthy individuals (6 individuals per subtype of HPC source). We identified 43 genes and 36 miRNAs differentially expressed in the various CD34⁺ cell sources. We observed that CD34⁺ cells from CB and BM showed similar gene and miRNA expression profiles, whereas CD34⁺ cells from G-PB had a very different expression pattern. Remarkably, 20 of the differentially expressed genes are targets of the differentially expressed miRNAs. Of note, the majority of genes differentially expressed in CD34⁺ cells from G-PB are involved in cell cycle regulation, promoting the process of proliferation, survival, hematopoiesis, and cell signaling, and are targets of overexpressed and underexpressed miRNAs in CD34⁺ cells from the same source. These data suggest significant differences in gene and miRNA expression among the various HPC sources used in transplantation. We hypothesize that the differentially expressed genes and miRNAs involved in cell cycle and proliferation might explain the differing kinetics of engraftment observed after transplantation of hematopoietic stem cells obtained from these different sources.     

T cell-expressed microRNAs critically regulate germinal center T follicular helper cell function and maintenance in acute viral infection in mice

Constitutive T cell-intrinsic miRNA expression is required for the differentiation of naïve CD4⁺ T cells into Tfh cells, thus making it difficult to study the role of miRNAs in the maintenance of already established Tfh cells and ongoing germinal center (GC) responses. To overcome this problem, we here used temporally controlled ablation of mature miRNAs specifically in CD4⁺ T cells during acute LCMV infection in mice. T cell-intrinsic miRNA expression was not only critical at early stages of Tfh cell differentiation, but also important for the maintenance of already established Tfh cells. In addition, CD4⁺ T cell-specific ablation of miRNAs resulted in impaired GC B cell responses. Notably, miRNA deficiency also compromised the antigen-specific CD4⁺ T cell compartment, Th1 cells, Treg cells, and Tfr cells. In conclusion, our results highlight miRNAs as important regulators of Tfh cells, thus providing novel insights into the molecular events that govern T cell–B cell interactions and Th cell identity.     

Validation of a flow cytometry based chemokine internalization assay for use in evaluating the pharmacodynamic response to a receptor antagonist

Background

Mobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells mobilized by each agent may contain different subtypes and have different functions. To characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects, AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects.

Results

Hierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92 cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples according to mobilization and isolation protocol and cell differentiation status.

Conclusion

HSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene expression microarrays maybe useful for assessing differences in HSCs.     

microRNA-200c overexpression inhibits chemoresistance,invasion and colony formation of human pancreatic cancer stem cells

Introduction: Cancer stem cells (CSCs) are believed to be ‘seed cell’ in cancer recurrence and metastasis. MicroRNAs (miRNAs) have emerged as potential therapeutic candidates due to their ability to regulate multiple targets involved in tumor progression and chemoresistance. The goal of this study was to investigate the role of miRNA-200c (miR-200c) in regulating colony formation, invasion and chemoresistance of human pancreatic cancer stem cells (PCSCs). Methods: PCSCs with CD24⁺CD44⁺ESA⁺ as the marker was sorted from PANC-1 cell line by fluorescence activated cell sorter (FACS). Quantitative real-time PCR (qRT-PCR) assay was used to detect the expression of miR-200c in PCSCs and PANC-1 cells. Transfection of miR-200c mimic into PCSCs was performed to establish miR-200c over-expressed cells. The effects of overexpressing miR-200c on PCSCs were examined by cell colony forming, invasion and survival assays in vitro. Results: Our data showed that CD24⁺CD44⁺ESA⁺ PCSCs (0.5%) were isolated from PANC-1 cells. Expression of miR-200c was significantly reduced in PCSCs compared with PANC-1 cells. In addition, the capability of colony formation, invasion and chemoresistance were markedly increased in PCSCs than that in PANC-1 cells. Adverse results were obtained in miR-200c overexpressing PCSCs transfected with miR-200c mimic. Conclusion: Our study demonstrated that miR-200c overexpression could decrease colony formation, invasion and chemoresistance of PCSCs. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer.     

Regulation of Toll-like receptor-mediated inflammatory response by microRNA-152-3p-mediated demethylation of MyD88 in systemic lupus erythematosus

Objective

microRNAs (miRNAs) play critical roles in embryogenesis, cell differentiation and the pathogenesis of several human diseases, including systemic lupus erythematosus (SLE). Toll-like receptors (TLRs) are also known to exert crucial functions in the immune response activation occurring in the pathogenesis of autoimmune diseases like SLE. Herein, the current study aimed to explore the potential role of miR-152-3p in TLR-mediated inflammatory response in SLE.

Methods

We determined the miR-152-3p expression profiles in CD4⁺ T cells and peripheral blood mononuclear cells (PBMCs) harvested from patients with SLE and healthy controls, and analyzed the correlation between miR-152-3p expression and clinicopathological parameters. CD70 and CD40L expression patterns in CD4⁺ T cells were assessed by RT-qPCR and flow cytometry. ChIP was adopted to determine the enrichment of DNA methyltransferase 1 (DNMT1) in the promoter region of myeloid differentiation factor 88 (MyD88).

Results

The obtained findings revealed that miR-152-3p was highly-expressed in CD4⁺ T cells and PBMCs of patients with SLE, and this high expression was associated with facial erythema, joint pain, double-stranded DNA, and IgG antibody. DNMT1 could be enriched in the MyD88 promoter, and miR-152-3p inhibited the methylation of MyD88 by targeting DNMT1. We also found that silencing miR-152-3p inhibited MyD88 expression not only to repress the autoreactivity of CD4⁺ T cells and but also to restrain their cellular inflammation, which were also validated in vivo.

Conclusion

Our study suggests that miR-152-3p promotes TLR-mediated inflammatory response in CD4⁺ T cells by regulating the DNMT1/MyD88 signaling pathway, which highlights novel anti-inflammatory target for SLE treatment.

     

The intermediate filament protein synemin (SYNM) was found to be more widespread in CD117+ gastrointestinal stromal cell tumors (GIST) than the CD34 transmembrane phosphoglycoprotein: an immunohistochemical study

Interstitial cells of Cajal (ICC) are the pacemaker cells in the gastrointestinal system, initiating the rhythmic systematic contraction of smooth muscle to regulate peristalsis. Benign ICC are identified immunohistochemically by positive staining for CD117, c-kit tyrosine kinase receptor. CD117 expression is maintained during the malignant transformation of ICC into gastrointestinal stromal cell tumors (GIST). However, to date, no single reliable marker for GIST has been identified by immunohistochemical (IHC) staining, and correct diagnosis depends on IHC staining results using multiple markers. This study compared the reactivities of synemin (SYNM) intermediate filament protein and CD34, a known marker for stem cells, endothelial cells, and many GIST tumors, in 54 formalin-fixed, paraffin-embedded GIST, which were determined by this study to be CD117 positive. Double-immunofluorescence (IF) staining was also used to assess whether CD117 and SYNM were co-expressed by ICC. While 81.5% of the GIST were CD34⁺, 92.9% of these tumors were SYNM⁺. ICC were CD117⁺/SYNM⁺, whereas mast cells were CD117⁺/SYNM^?. Because the percentage of CD117⁺/SYNM⁺ GIST was higher than the percentage of 117⁺/CD34⁺ GIST, this study suggested that SYNM was a better marker than CD34 for GIST diagnosis. In addition, differential expression of SYNM and CD117 helped distinguish between ICC and mast cells.     

Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor-matched CD133(+) cells with the more differentiated CD34(+) CD133(-) and CD34(-) CD133(-) cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR-142-3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34(+) CD133(-) cells. Eighteen miRNAs were significantly differentially expressed between CD133(+) and CD34(+) CD133(-) cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase-based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR-142-3p as well as tropomyosin 1 and frizzled homolog 5 by miR-29a. Overexpression of miRNAs in CD133(+) cells demonstrated that miR-142-3p has a negative influence on the overall colony-forming ability. In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.     

Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-β receptor type II mouse

Dominant-negative TGF-β receptor II (dnTGF-βRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-β receptor is expressed by both CD4⁺ and CD8⁺ T cells and leads to greatly reduced (but not absent) TGF-β signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-β signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-β signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8⁺ T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-βRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-βRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-βRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC.     

Reaction of CD117+ Cells to Renal Lesion under Conditions of Phagocytic Mononuclear System Stimulation

The content of CD117⁺ cells in the kidneys and CD45^lowCD117⁺ cells in the bone marrow and blood of mice were studied after partial nephrectomy and under conditions of macrophage stimulation with 3-aminophthalhydrazide. The counts of tubular CD117⁺ epitheliocytes sharply increased and the content of CD45^lowCD117⁺ cells in the bone marrow decreased after renal damage. Injection of 3-aminophthalhydrazide stimulated the expression of CD117 by renal epitheliocytes and led to reduction of CD45^lowCD117⁺ cell counts in the bone marrow and blood. Macrophages stimulated proliferative processes in the kidney and differentiation of stem cells in the bone marrow due to synergic effects of their cytokines and stem cell factor.     

Systematic identification of microRNA and messenger RNA profiles in hepatitis C virus-infected human hepatoma cells

In order to investigate the global and dynamic host microRNAs (miRNAs)/messenger RNAs (mRNAs) expression alteration during in vitro acute HCV infection, a comprehensive microarray analysis was performed using human hepatoma cells. Totally, 108 human miRNAs and 1247 mRNAs were identified whose expression levels changed for more than 2.0-fold in response to HCV infection. Upon HCV infection, signature from the unique miRNA expression pattern reflected the involvement of miRNA-regulated host cellular physiology and antiviral mechanism, whereas a preponderance of differentially regulated genes associated with metabolism, cell growth, apoptosis and cytokine/chemokine pathways. Furthermore, a reverse regulatory association of differentially expressed miRNAs and their predicted targets was constructed. Finally, the differentially expressed miRNAs such as miR-24, miR-149?, miR-638 and miR-1181 were identified to be involved in HCV entry, replication and propagation. These results suggest that combined miRNA and mRNA profiling may have superior potential as a diagnostic and mechanistic feature in HCV infection.     

miR-451 limits CD4<Superscript>+</Superscript> T cell proliferative responses to infection in mice

MicroRNAs (miRNAs) are major regulators of cell responses, particularly in stressed cell states and host immune responses. Some miRNAs have a role in pathogen defense, including regulation of immune responses to Plasmodium parasite infection. Using a nonlethal mouse model of blood stage malaria infection, we have found that miR-451^?/? mice infected with Plasmodium yoelii XNL cleared infection at a faster rate than did wild-type (WT) mice. MiR-451^?/? mice had an increased leukocyte response to infection, with the protective phenotype primarily driven by CD4⁺ T cells. WT and miR-451^?/? CD4⁺ T cells had similar activation responses, but miR-451^?/? CD4⁺ cells had significantly increased proliferation, both in vitro and in vivo. Myc is a miR-451 target with a central role in cell cycle progression and cell proliferation. CD4⁺ T cells from miR-451^?/? mice had increased postactivation Myc expression. RNA-Seq analysis of CD4⁺ cells demonstrated over 5000 differentially expressed genes in miR-451^?/? mice postinfection, many of which are directly or indirectly Myc regulated. This study demonstrates that miR-451 regulates T cell proliferative responses in part via a Myc-dependent mechanism.     

Inhibition of cancer stem cell-like properties and reduced chemoradioresistance of glioblastoma using microRNA145 with cationic polyurethane-short branch PEI

Glioblastomas (GBMs) are the most common primary brain tumors with poor prognosis. CD133 has been considered a putative marker of cancer stem cells (CSCs) in malignant cancers, including GBMs. MicroRNAs (miRNAs), highly conserved small RNA molecules, may target oncogenes and have potential as a therapeutic strategy against cancer. However, the role of miRNAs in GBM-associated CSCs remains mostly unclear. In this study, our miRNA/mRNA-microarray and RT-PCR analysis showed that the expression of miR145 (a tumor-suppressive miRNA) is inversely correlated with the levels of Oct4 and Sox2 in GBM-CD133⁺ cells and malignant glioma specimens. We demonstrated that miR145 negatively regulates GBM tumorigenesis by targeting Oct4 and Sox2 in GBM-CD133⁺. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic-delivery vehicle, PU-PEI-mediated miR145 delivery to GBM-CD133⁺ significantly inhibited their tumorigenic and CSC-like abilities and facilitated their differentiation into CD133⁻-non-CSCs. Furthermore, PU-PEI-miR145-treated GBM-CD133⁺ effectively suppressed the expression of drug-resistance and anti-apoptotic genes and increased the sensitivity of the cells to radiation and temozolomide. Finally, the in vivo delivery of PU-PEI-miR145 alone significantly suppressed tumorigenesis with stemness, and synergistically improved the survival rate when used in combination with radiotherapy and temozolomide in orthotopic GBM-CD133⁺-transplanted immunocompromised mice. Therefore, PU-PEI-miR145 is a novel therapeutic approach for malignant brain tumors.     

MicroRNA Microarray Expression Profiling in Human Myocardial Infarction

MicroRNAs (miRNAs), small non-coding RNA molecules, are negative regulators of gene expression. Recent studies have indicated their role in various forms of cardiovascular disease. In spite of the number of miRNA microarray analyses performed, little is known about the genome-wide miRNA expression pattern in human myocardial infarction (MI). Using miRNA microarrays and bioinformatic analysis, miRNA expression was analyzed on human MI and foetal hearts compared to healthy adult hearts, to determine whether there is any similar expression pattern between MI and foetal hearts, and to identified miRNAs that have not previously been described as dysregulated in cardiovascular diseases. Of 719 miRNAs analyzed, ∼ 50% were expressed in human hearts, 77 miRNAs were absent from all tested tissues and 57 were confidently dysregulated in at least one tested group. Some expression patterns appeared to be similar in MI and foetal hearts. Bioinformatic analysis revealed 10 miRNAs as dysregulated in MI not yet related to cardiovascular disease, and 5 miRNAs previously described only in animal models of cardiovascular diseases. Finally, qRT-PCR analysis confirmed dysregulation of 7 miRNAs, miR-150, miR-186, miR-210, miR-451, and muscle-specific, miR-1 and miR-133a/b; all of these are believed to be involved in various physiological and pathological processes.     

Altered microRNA Expression and Immunosuppressive Cytokine Production by Regulatory T Cells of Ulcerative Colitis Patients

Regulatory T (Treg) cells are essential for maintenance of peripheral tolerance and prevention of autoimmune diseases in part by producing immunosuppressive cytokines. Recently, microRNAs (miRNAs) have also been involved in autoimmune disorders, not least for their crucial role in the regulation of Treg biology and function. We simultaneously investigated the concentration of IL-35, IL-10, TGF-β, and sCD25 in supernatant of cell culture and the expression patterns of several miRNAs in CD4⁺CD25⁺ CD127^?/low FoxP3⁺ Tregs of ulcerative colitis (UC) patients. Significantly lower levels of IL-10 and IL-35 were observed in Treg cultures of UC patients. miR-21, miR-146a, and miR-155 levels were downregulated and miR-31 level was upregulated in Tregs of patients. Our results suggest that microRNAs may serve as a novel regulator in function and homoeostasis of UC Treg cells, providing a key role for them in pathophysiology of UC.     

Distinctive microRNA expression profiles in CD34+ bone marrow cells from patients with myelodysplastic syndrome

MicroRNAs (miRNAs) are small non-coding RNAs functioning as regulators of hematopoiesis. Their differential expression patterns have been linked with various pathological processes originating from hematopoietic stem cells (HSCs). However, limited information is available regarding the role of miRNAs in myelodysplastic syndrome (MDS). Using miRNA arrays, we measured expression of 1,145 miRNAs in CD34+ bone marrow cells obtained from 39 MDS and acute myeloid leukemia (AML) evolved from MDS patients, and compared them with those of six healthy donors. Differential miRNA expression was analyzed and a panel of upregulated (n=13) and downregulated (n=9) miRNAs were found (P<0.001) in MDS/AML patients. An increased expression of a large miRNA cluster mapped within the 14q32 locus was detected. Differences in miRNA expression of MDS subtypes showed a distinction between early and advanced MDS; an apparent dissimilarity was observed between RAEB-1 and RAEB-2 subtypes. In early MDS, we monitored upregulation of proapoptotic miR-34a, which may contribute to the increased apoptosis of HSCs. Patients with 5q deletion were characterized by decreased levels of miR-143(*) and miR-378 mapped within the commonly deleted region at 5q32. This is an early report describing differential expression in MDS CD34+ cells, likely reflecting their disease-specific regulation.