PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1311542)

Clipboard (0)
None

Related Articles

1.  Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients 
Arthritis Research & Therapy  2008;10(4):R101.
Introduction
MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.
Methods
Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.
Results
Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.
Conclusions
Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.
doi:10.1186/ar2493
PMCID: PMC2575615  PMID: 18759964
2.  Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis 
Introduction
MicroRNAs (miRNAs), endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes, are present in human plasma in a stable form. In this study, we investigated whether miRNAs are also stably present in synovial fluids and whether plasma and synovial fluid miRNAs could be biomarkers of rheumatoid arthritis (RA) and osteoarthritis (OA).
Methods
We measured concentrations of miR-16, miR-132, miR-146a, miR-155 and miR-223 in synovial fluid from patients with RA and OA, and those in plasma from RA, OA and healthy controls (HCs) by quantitative reverse transcription-polymerase chain reaction. Furthermore, miRNAs in the conditioned medium of synovial tissues, monolayer fibroblast-like synoviocytes, and mononuclear cells were examined. Correlations between miRNAs and biomarkers or disease activities of RA were statistically examined.
Results
Synovial fluid miRNAs were present and as stable as plasma miRNAs for storage at -20°C and freeze-thawing from -20°C to 4°C. In RA and OA, synovial fluid concentrations of miR-16, miR-132, miR-146a, and miR-223 were significantly lower than their plasma concentrations, and there were no correlation between plasma and synovial fluid miRNAs. Interestingly, synovial tissues, fibroblast-like synoviocytes, and mononuclear cells secreted miRNAs in distinct patterns. The expression patterns of miRNAs in synovial fluid of OA were similar to miRNAs secreted by synovial tissues. Synovial fluid miRNAs of RA were likely to originate from synovial tissues and infiltrating cells. Plasma miR-132 of HC was significantly higher than that of RA or OA with high diagnosability. Synovial fluid concentrations of miR-16, miR-146a miR-155 and miR-223 of RA were significantly higher than those of OA. Plasma miRNAs or ratio of synovial fluid miRNAs to plasma miRNAs, including miR-16 and miR-146a, significantly correlated with tender joint counts and 28-joint Disease Activity Score.
Conclusions
Plasma miRNAs had distinct patterns from synovial fluid miRNAs, which appeared to originate from synovial tissue. Plasma miR-132 well differentiated HCs from patients with RA or OA, while synovial fluid miRNAs differentiated RA and OA. Furthermore, plasma miRNAs correlated with the disease activities of RA. Thus, synovial fluid and plasma miRNAs have potential as diagnostic biomarkers for RA and OA and as a tool for the analysis of their pathogenesis.
doi:10.1186/ar3013
PMCID: PMC2911870  PMID: 20470394
3.  Altered Expression of MicroRNA-203 in Rheumatoid Arthritis Synovial Fibroblasts and Its Role in Fibroblast Activation 
Arthritis and rheumatism  2011;63(2):373-381.
Objective
MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR-155 and miR-146a in rheumatoid arthritis (RA) and showed a repressive effect of miR-155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR-203 in RASFs and analyze its role in fibroblast activation.
Methods
Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real-time polymerase chain reaction (PCR)–based screening of 260 individual miRNA. Transfection of miR-203 precursor was used to analyze the function of miR-203 in RASFs. Levels of interleukin-6 (IL-6) and MMPs were measured by real-time PCR and enzyme-linked immunosorbent assay. RASFs were stimulated with IL-1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5-azacytidine (5-azaC). Activity of IκB kinase 2 was inhibited with SC-514.
Results
Expression of miR-203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR-203 did not change upon stimulation with IL-1β, TNFα, or LPS; however, DNA demethylation with 5-azaC increased the expression of miR-203. Enforced expression of miR-203 led to significantly increased levels of MMP-1 and IL-6. Induction of IL-6 by miR-203 overexpression was inhibited by blocking of the NF-κB pathway. Basal expression levels of IL-6 correlated with basal expression levels of miR-203.
Conclusion
The current results demonstrate methylation-dependent regulation of miR-203 expression in RASFs. Importantly, they also show that elevated levels of miR-203 lead to increased secretion of MMP-1 and IL-6 via the NF-κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.
doi:10.1002/art.30115
PMCID: PMC3116142  PMID: 21279994
4.  Expression of microRNA-146 in osteoarthritis cartilage 
Arthritis and rheumatism  2009;60(4):1035-1041.
Objective
A role of microRNAs, which are ∼22- nucleotide non coding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in cartilage from patients with osteoarthritis (OA).
Methods
The expression of miR-146 in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146 by cultures of normal human articular chondrocytes following stimulation with interleukin-1β (IL-1β) was examined by quantitative RT-PCR.
Results
All cartilage samples were divided into three groups according to a modified Mankin scale; grade I: 0 - 5, grade II: 6 - 10, grade III: 11 - 14. In OA cartilage samples of grade I, the expression of miR-146a and Col2a1 was significantly higher than that of other groups (p<0.05). In OA cartilage of grades II and III, the expression of miR-146a and Col2a1 decreased while the expression of MMP13 was elevated in grade II. These data show that miR-146a is expressed intensely in cartilage with a low Mankin grade, and that miR-146a expression decreases in accordance with level of MMP13 expression. Section in situ hybridization of pri-miR-146a revealed that pri-miR-146a is expressed in chondrocytes in all layers, especially in the superficial layer where it is intensely expressed. The expression of miR-146 was markedly elevated by IL-1β stimulation in human chondrocytes in vitro.
Conclusion
This study shows that miR-146 is intensely expressed in low grade OA cartilage, and that its expression is induced by stimulation of IL-1β. MiR-146 might play a role in OA cartilage pathogenesis.
doi:10.1002/art.24404
PMCID: PMC2670476  PMID: 19333945
5.  Altered microRNA expression profile with miR-146a upregulation in CD4+ T cells from patients with rheumatoid arthritis 
Introduction
Increasing evidence indicates that microRNAs (miRNAs) play a critical role in the pathogenesis of inflammatory diseases. The aim of the study was to investigate the expression pattern and function of miRNAs in CD4+ T cells from patients with rheumatoid arthritis (RA).
Methods
The expression profile of miRNAs in CD4+ T cells from synovial fluid (SF) and peripheral blood of 33 RA patients was determined by microarray assay and validated by qRT-PCR analysis. The correlation between altered expression of miRNAs and cytokine levels was determined by linear regression analysis. The role of miR-146a overexpression in regulating T cell apoptosis was evaluated by flow cytometry. A genome-wide gene expression analysis was further performed to identify miR-146a-regulated genes in T cells.
Results
miRNA expression profile analysis revealed that miR-146a expression was significantly upregulated while miR-363 and miR-498 were downregulated in CD4+ T cells of RA patients. The level of miR-146a expression was positively correlated with levels of tumor necrosis factor-alpha (TNF-α), and in vitro studies showed TNF-α upregulated miR-146a expression in T cells. Moreover, miR-146a overexpression was found to suppress Jurkat T cell apoptosis. Finally, transcriptome analysis of miR-146a overexpression in T cells identified Fas associated factor 1 (FAF1) as a miR-146a-regulated gene, which was critically involved in modulating T cell apoptosis.
Conclusions
We have detected increased miR-146a in CD4+ T cells of RA patients and its close correlation with TNF-α levels. Our findings that miR-146a overexpression suppresses T cell apoptosis indicate a role of miR-146a in RA pathogenesis and provide potential novel therapeutic targets.
doi:10.1186/ar3006
PMCID: PMC2911863  PMID: 20459811
6.  MicroRNA-146a expresses in interleukin-17 producing T cells in rheumatoid arthritis patients 
Background
Interleukin (IL)-17 is an important factor in rheumatoid arthritis (RA) pathogenesis. MicroRNA (miRNA)s are a family of non coding RNAs and associated with human diseases including RA. The purpose of this study is to identify the miRNAs in the differentiation of IL-17 producing cells, and analyze their expression pattern in the peripheral blood mononuclear cells (PBMC) and synovium from RA patients.
Methods
IL-17 producing cells were expanded from CD4+T cell. MiRNA microarray was performed to identify the miRNAs in the differentiation of IL-17 producing cells. Quantitative polymerase chain reaction was performed to examine the expression patterns of the identified miRNAs in the PBMC and synovium from RA and osteoarthritis (OA) patients. Double staining combining in situ hybridization and immunohistochemistry of IL-17 was performed to analyze the expression pattern of identified miRNA in the synovium.
Results
Six miRNAs, let-7a, miR-26, miR-146a/b, miR-150, and miR-155 were significantly up regulated in the IL-17 producing T cells. The expression of miR-146a and IL-17 was higher than in PBMC in the patients with low score of Larsen grade and short disease duration. MiR-146a intensely expressed in RA synovium in comparison to OA. MiR-146a expressed intensely in the synovium with hyperplasia and high expression of IL-17 from the patients with high disease activity. Double staining revealed that miR-146a expressed in IL-17 expressing cells.
Conclusion
These results indicated that miR-146a was associated with IL-17 expression in the PBMC and synovium in RA patients. There is the possibility that miR-146a participates in the IL-17 expression.
doi:10.1186/1471-2474-11-209
PMCID: PMC2950393  PMID: 20840794
7.  MicroRNA-146a Provides Feedback Regulation of Lyme Arthritis but Not Carditis during Infection with Borrelia burgdorferi 
PLoS Pathogens  2014;10(6):e1004212.
MicroRNAs have been shown to be important regulators of inflammatory and immune responses and are implicated in several immune disorders including systemic lupus erythematosus and rheumatoid arthritis, but their role in Lyme borreliosis remains unknown. We performed a microarray screen for expression of miRNAs in joint tissue from three mouse strains infected with Borrelia burgdorferi. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling, in all three strains, suggesting it plays an important role in the in vivo response to B. burgdorferi. Infection of B6 miR-146a−/− mice with B. burgdorferi revealed a critical nonredundant role of miR-146a in modulating Lyme arthritis without compromising host immune response or heart inflammation. The impact of miR-146a was specifically localized to the joint, and did not impact lesion development or inflammation in the heart. Furthermore, B6 miR-146a−/− mice had elevated levels of NF-κB-regulated products in joint tissue and serum late in infection. Flow cytometry analysis of various lineages isolated from infected joint tissue of mice showed that myeloid cell infiltration was significantly greater in B6 miR-146a−/− mice, compared to B6, during B. burgdorferi infection. Using bone marrow-derived macrophages, we found that TRAF6, a known target of miR-146a involved in NF-κB activation, was dysregulated in resting and B. burgdorferi-stimulated B6 miR-146a−/− macrophages, and corresponded to elevated IL-1β, IL-6 and CXCL1 production. This dysregulated protein production was also observed in macrophages treated with IL-10 prior to B. burgdorferi stimulation. Peritoneal macrophages from B6 miR-146a−/− mice also showed enhanced phagocytosis of B. burgdorferi. Together, these data show that miR-146a-mediated regulation of TRAF6 and NF-κB, and downstream targets such as IL-1β, IL-6 and CXCL1, are critical for modulation of Lyme arthritis during chronic infection with B. burgdorferi.
Author Summary
Lyme Disease is caused by infection with the bacteria Borrelia burgdorferi, is transmitted through infected deer ticks (Ixodes scapularis), and often leads to arthritis that can persist, even after antibiotic treatment. Here, we have identified a microRNA that is critical in modulating Lyme arthritis, but not carditis. This microRNA, miR-146a, is a negative regulator of NF-κB signaling, known to be important in host defense against pathogens, and long suspected to play a role in Lyme arthritis development. Mice lacking miR-146a develop more severe arthritis and show signs of hyperactive NF-κB activation during the persistent phase of infection. Heart manifestations of disease were not altered. Furthermore, this severe arthritis is independent of host defense, since these mice are better able to clear invading bacteria in joints, and bacterial numbers are similar in heart and ear tissue. We identified TRAF6 as an important target of miR-146a-mediated NF-κB regulation of pro-inflammatory cytokines IL-6 and IL-1β, as well as chemokines CXCL1 and CXCL2. Our data demonstrate the importance of maintaining appropriate regulation of amplitude and resolution of NF-κB activation during Borrelia burgdorferi infection, and provide a novel model for elucidating the role of NF-κB in Lyme arthritis development, independent of effect on host defense.
doi:10.1371/journal.ppat.1004212
PMCID: PMC4072785  PMID: 24967703
8.  Acute-phase serum amyloid A production by rheumatoid arthritis synovial tissue 
Arthritis Research  2000;2(2):142-144.
Acute-phase serum amyloid A (A-SAA) is a major component of the acute-phase response. A sustained acute-phase response in rheumatoid arthritis (RA) is associated with increased joint damage. A-SAA mRNA expression was confirmed in all samples obtained from patients with RA, but not in normal synovium. A-SAA mRNA expression was also demonstrated in cultured RA synoviocytes. A-SAA protein was identified in the supernatants of primary synoviocyte cultures, and its expression colocalized with sites of macrophage accumulation and with some vascular endothelial cells. It is concluded that A-SAA is produced by inflamed RA synovial tissue. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Introduction:
Serum amyloid A (SAA) is the circulating precursor of amyloid A protein, the fibrillar component of amyloid deposits. In humans, four SAA genes have been described. Two genes (SAA1 and SAA2) encode A-SAA and are coordinately induced in response to inflammation. SAA1 and SAA2 are 95% homologous in both coding and noncoding regions. SAA3 is a pseudogene. SAA4 encodes constitutive SAA and is minimally inducible. A-SAA increases dramatically during acute inflammation and may reach levels that are 1000-fold greater than normal. A-SAA is mainly synthesized in the liver, but extrahepatic production has been demonstrated in many species, including humans. A-SAA mRNA is expressed in RA synoviocytes and in monocyte/macrophage cell lines such as THP-1 cells, in endothelial cells and in smooth muscle cells of atherosclerotic lesions. A-SAA has also been localized to a wide range of histologically normal tissues, including breast, stomach, intestine, pancreas, kidney, lung, tonsil, thyroid, pituitary, placenta, skin and brain.
Aims:
To identify the cell types that produce A-SAA mRNA and protein, and their location in RA synovium.
Materials and methods:
Rheumatoid synovial tissue was obtained from eight patients undergoing arthroscopic biopsy and at joint replacement surgery. Total RNA was analyzed by reverse transcription (RT) polymerase chain reaction (PCR) for A-SAA mRNA. PCR products generated were confirmed by Southern blot analysis using human A-SAA cDNA. Localization of A-SAA production was examined by immunohistochemistry using a rabbit antihuman A-SAA polyclonal antibody. PrimaryRA synoviocytes were cultured to examine endogenous A-SAA mRNA expression and protein production.
Results:
A-SAA mRNA expression was detected using RT-PCR in all eight synovial tissue samples studied. Figure 1 demonstrates RT-PCR products generated using synovial tissue from three representative RA patients. Analysis of RA synovial tissue revealed differences in A-SAA mRNA levels between individual RA patients.
In order to identify the cells that expressed A-SAA mRNA in RA synovial tissue, we analyzed primary human synoviocytes (n = 2). RT-PCR analysis revealed A-SAA mRNA expression in primary RA synoviocytes (n = 2; Fig. 2). The endogenous A-SAA mRNA levels detected in individual primary RA synoviocytes varied between patients. These findings are consistent with A-SAA expression in RA synovial tissue (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were relatively similar in the RA synoviocytes examined (Fig. 2). A-SAA protein in the supernatants of primary synoviocyte cultures from four RA patients was measured using ELISA. Mean values of a control and four RA samples were 77.85, 162.5, 249.8, 321.5 and 339.04 μg/l A-SAA, respectively, confirming the production of A-SAA protein by the primary RA synoviocytes. Immunohistochemical analysis was performed to localize sites of A-SAA production in RA synovial tissue. Positive staining was present in both the lining and sublining layers of all eight RA tissues examined (Fig. 3a). Staining was intense and most prominent in the cells closest to the surface of the synovial lining layer. Positively stained cells were evident in the perivascular areas of the sublining layer. In serial sections stained with anti-CD68 monoclonal antibody, positive staining of macrophages appeared to colocalize with A-SAA-positive cells (Fig. 3b). Immunohistochemical studies of cultured primary RA synoviocytes confirmed specific cytoplasmic A-SAA expression in these cells. The specificity of the staining was confirmed by the absence of staining found on serial sections and synoviocyte cells treated with IgG (Fig. 3c).
Discussion:
This study demonstrates that A-SAA mRNA is expressed in several cell populations infiltrating RA synovial tissue. A-SAA mRNA expression was observed in all eight unseparated RA tissue samples studied. A-SAA mRNA expression and protein production was demonstrated in primary cultures of purified RA synoviocytes. Using immunohistochemical techniques, A-SAA protein appeared to colocalize with both lining layer and sublining layer synoviocytes, macrophages and some endothelial cells. The detection of A-SAA protein in culture media supernatants harvested from unstimulated synoviocytes confirms endogenous A-SAA production, and is consistent with A-SAA mRNA expression and translation by the same cells. Moreover, the demonstration of A-SAA protein in RA synovial tissue, RA cultured synoviocytes, macrophages and endothelial cells is consistent with previous studies that demonstrated A-SAA production by a variety of human cell populations.
The RA synovial lining layer is composed of activated macrophages and fibroblast-like synoviocytes. The macrophage is the predominant cell type and it has been shown to accumulate preferentially in the surface of the lining layer and in the perivascular areas of the sublining layer. Nevertheless, our observations strongly suggest that A-SAA is produced not only by synoviocytes, but also by synovial tissue macrophage populations. Local A-SAA protein production by vascular endothelial cells was detected in some, but not all, of the tissues examined. The reason for the variability in vascular A-SAA staining is unknown, but may be due to differences in endothelial cell activation, events related to angiogenesis or the intensity of local inflammation.
The value of measuring serum A-SAA levels as a reliable surrogate marker of inflammation has been demonstrated for several diseases including RA, juvenile chronic arthritis, psoriatic arthropathy, ankylosing spondylitis, Behçet's disease, reactive arthritis and Crohn's disease. It has been suggested that serum A-SAA levels may represent the most sensitive measurement of the acute-phase reaction. In RA, A-SAA levels provide the strongest correlations with clinical measurements of disease activity, and changes in serum levels best reflect the clinical course.
A number of biologic activities have been described for A-SAA, including several that are relevant to the understanding of inflammatory and tissue-degrading mechanisms in human arthritis. A-SAA induces migration, adhesion and tissue infiltration of circulating monocytes and polymorphonuclear leukocytes. In addition, human A-SAA can induce interleukin-1β, interleukin-1 receptor antagonist and soluble type II tumour necrosis factor receptor production by a monocyte cell line. Moreover, A-SAA can stimulate the production of cartilage-degrading proteases by both human and rabbit synoviocytes. The effects of A-SAA on protease production are interesting, because in RA a sustained acute-phase reaction has been strongly associated with progressive joint damage. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Conclusion:
In contrast to noninflamed synovium, A-SAA mRNA expression was identified in all RA tissues examined. A-SAA appeared to be produced by synovial tissue synoviocytes, macrophages and endothelial cells. The observation of A-SAA mRNA expression in cultured RA synoviocytes and human RA synovial tissue confirms and extends recently published findings that demonstrated A-SAA mRNA expression in stimulated RA synoviocytes, but not in unstimulated RA synoviocytes.
PMCID: PMC17807  PMID: 11062604
acute-phase response; rheumatoid arthritis; serum amyloid A; synovial tissue
9.  MicroRNAs expression signatures are associated with lineage and survival in acute leukemias 
Blood cells, molecules & diseases  2010;44(3):191-197.
MicroRNAs (miRNAs) are small (~22 nucleotide) non-coding RNAs whose altered expression has been associated with various types of cancers, including leukemia. In the present study, we conducted a quantitative PCR (qPCR) analysis of expression of 23 human precursor miRNAs in bone marrow specimens of 85 Chinese primary leukemia patients, including 53 acute myeloid leukemia (AML) and 32 acute lymphoblastic leukemia (ALL) cases. We show that 16 miRNAs were differentially expressed between AMLs and ALLs; Of them, eight were previously reported (i.e., miR-23a, miR-27a/b, miR-128a, miR-128b, miR-221, miR-222, miR-223, and let-7b) and eight were newly identified (i.e., miR-17, miR-20a, miR-29a/c, miR-29b, miR-146a, miR-150, miR-155, and miR-196b). More importantly, through correlating miRNA expression signatures with outcome of patients, we further show that expression signatures of a group of miRNAs are associated with overall survival of patients. Of them, three (i.e., miR-146a, miR-181a/c, and miR-221), five (i.e., miR-25, miR-26a, miR-29b, miR-146a, and miR-196b), and three (i.e., miR-26a, miR-29b, and miR-146a) miRNAs are significantly associated with overall survival (P<0.05) of the 32 ALL, 53 AML, and 40 non-M3 AML patients, respectively. Particularly, the expression signature of miR-146a is significantly inversely correlated with overall survival of both ALL and AML patients. The prognostic significance of miR-146a in AML has been confirmed further in an independent study of 61 Chinese new AML patient samples. We also identified 622 putative target genes of miR-146a that are predicted by at least three out of the five major prediction programs (i.e., TragetScan, PicTar, miRanda, miRBase Targets, and PITA); Through gene ontology analysis, we found that these genes were particularly enriched (2–9 fold higher than expected by chance) in the GO categories of “negative regulation of biology processes”, “negative regulation of cellular processes”, “apoptosis”, and “cell cycle”, which may be related to the association of miR-146a with poor survival.
doi:10.1016/j.bcmd.2009.12.010
PMCID: PMC2829339  PMID: 20110180
microRNA; acute leukemia; lineage; survival analysis; miR-146a
10.  MicroRNA-146a: A Key Regulator of Astrocyte-Mediated Inflammatory Response 
PLoS ONE  2012;7(9):e44789.
Increasing evidence supports the involvement of microRNAs (miRNA) in the regulation of inflammation in human neurological disorders. In the present study we investigated the role of miR-146a, a key regulator of the innate immune response, in the modulation of astrocyte-mediated inflammation. Using Taqman PCR and in situ hybridization, we studied the expression of miR-146a in epilepsy-associated glioneuronal lesions which are characterized by prominent activation of the innate immune response. In addition, cultured human astrocytes were used to study the regulation of miR-146a expression in response to proinflammatory cytokines. qPCR and western blot were used to evaluate the effects of overexpression or knockdown of miR-146a on IL-1β signaling. Downstream signaling in the IL-1β pathway, as well as the expression of IL-6 and COX-2 were evaluated by western blot and ELISA. Release several cytokines was evaluated using a human magnetic multiplex cytokine assay on a Luminex® 100™/200™ platform. Increased expression of miR-146a was observed in glioneuronal lesions by Taqman PCR. MiR-146a expression in human glial cell cultures was strongly induced by IL-1β and blocked by IL-1β receptor antagonist. Modulation of miR-146a expression by transfection of astrocytes with anti-miR146a or mimic, regulated the mRNA expression levels of downstream targets of miR-146a (IRAK-1, IRAK-2 and TRAF-6) and the expression of IRAK-1 protein. In addition, the expression of IL-6 and COX-2 upon IL-1β stimulation was suppressed by increased levels of miR-146a and increased by the reduction of miR-146a. Modulation of miR-146a expression affected also the release of several cytokines such as IL-6 and TNF-α. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation and suggests that this miR may represent a novel target for therapeutic strategies.
doi:10.1371/journal.pone.0044789
PMCID: PMC3441440  PMID: 23028621
11.  Chikungunya Virus Exploits miR-146a to Regulate NF-κB Pathway in Human Synovial Fibroblasts 
PLoS ONE  2014;9(8):e103624.
Objectives
Chikungunya virus causes chronic infection with manifestations of joint pain. Human synovial fibroblasts get infected with CHIKV and could lead to pro-inflammatory responses. MicroRNAs have potentials to regulate the gene expression of various anti-viral and pro-inflammatory genes. The study aims to investigate the role of miR-146a in modulation of inflammatory responses of human synovial fibroblasts by Chikungunya virus.
Methods
To study the role of miR-146a in CHIKV pathogenesis in human synovial cells and underlying inflammatory manifestations, we performed CHIKV infection in primary human synovial fibroblasts. Western blotting, real-time PCR, luciferase reporter assay, overexpression and knockdown of cellular miR-146a strategies have been employed to validate the role of miR-146a in regulation of pro-inflammatory NF-κB pathway.
Results
CHIKV infection induced the expression of cellular miR-146a, which resulted into down-regulation of TRAF6, IRAK1, IRAK2 and increased replication of CHIKV in human synovial fibroblasts. Exogenous expression of miR-146a in human synovial fibroblasts led to decreased expression of TRAF6, IRAK1, IRAK2 and decreased replication of CHIKV. Inhibition of cellular miR-146a by anti-miR-146a restored the expression levels of TRAF6, IRAK1 and IRAK2. Downregulation of TRAF6, IRAK1 and IRAK2 led to downstream decreased NF-κB activation through negative feedback loop.
Conclusion
This study demonstrated the mechanism of exploitation of cellular miR-146a by CHIKV in modulating the host antiviral immune response in primary human synovial fibroblasts.
doi:10.1371/journal.pone.0103624
PMCID: PMC4118904  PMID: 25083878
12.  Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro 
MicroRNA (miR)-146a is known to be overexpressed in osteoarthritis (OA). However, the role of miR-146a in OA has not yet been fully elucidated. In the present study, we applied mechanical pressure of 10 MPa to human chondrocytes for 60 min in order to investigate the expression of miR-146a and apoptosis following the mechanical pressure injury. Normal human chondrocytes were transfected with an miR-146a mimic or an inhibitor to regulate miR-146a expression. Potential target genes of miR-146a were predicted using bioinformatics. Moreover, luciferase reporter assay confirmed that Smad4 was a direct target of miR-146a. The expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by quantitative reverse transcription PCR and/or western blot analysis. The effects of miR-146a on apoptosis were detected by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry. The results indicated that mechanical pressure affected chondrocyte viability and induced the early apoptosis of chondrocytes. Mechanical pressure injury increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in the chondrocytes. In the human chondrocytes, the upregulation of miR-146a induced apoptosis, upregulated VEGF expression and downregulated Smad4 expression. In addition, the knockdown of miR-146a reduced cell apoptosis, upregulated Smad4 expression and downregulated VEGF expression. Smad4 was identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3′-untranslated region (3′-UTR) of its mRNA. Furthermore, the upregulation of VEGF induced by miR-146a was mediated by Smad4 in the chondrocytes subjected to mechanical pressure injury. These results demonstrated that miR-146a was overexpressed in our chondrocyte model of experimentally induced human mechanical injury, accompanied by the upregulation of VEGF and the downregulation of Smad4 in vitro. Moreover, our data suggest that miR-146a is involved in human chondrocyte apoptosis in response to mechanical injury, and may contribute to the mechanical injury of chondrocytes, as well as to the pathogenesis of OA by increasing the levels of VEGF and damaging the transforming growth factor (TGF)-β signaling pathway through the targeted inhibition of Smad4 in cartilage.
doi:10.3892/ijmm.2014.1808
PMCID: PMC4094584  PMID: 24939082
apoptosis; osteoarthritis; mechanical injury; chondrocyte; miR-146a
13.  miR-146a promotes the initiation and progression of melanoma by activating Notch signaling 
eLife  2014;3:e01460.
Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma.
DOI: http://dx.doi.org/10.7554/eLife.01460.001
eLife digest
Cancer is a leading cause of death worldwide, and although cancer can have many causes, mutations to a small number of genes are often responsible for a large number of cancer cases. For example, about 70% of cases of the deadliest form of skin cancer have mutations in two specific genes. Many cancer-causing genes are regulated by small RNA molecules that impede the function of other genes by blocking the machinery that turns a gene into a functional protein. However, until recently there was a limited understanding of the role of these microRNAs in the skin cancers caused by the most common mutations.
Now, Forloni et al. have looked at all the microRNAs present in cells carrying a mutated form of the one of these genes, and compared these with the microRNAs found in healthy cells. A microRNA, called miR-146a, was discovered to be much more common in the cancer cells. Moreover, production of this microRNA was increased by the two cancer-causing mutations. Forloni et al. found that increasing production of this microRNA caused human skin cancer cells to grow faster, and also caused tumors to develop in mice. Reducing the level of miR-146a had the opposite effect.
Forloni et al. also looked at cancer cells taken from individuals at different stages of skin cancer and found that, as the disease progresses, an unprocessed form of miR-146a tends to acquire a mutation, which leads to much higher levels of the active, processed form of miR-146a in the cancer cells. High levels of miR-146a also switched off a gene that encodes for a protein that, in turn, switches off a protein called NOTCH that is linked to skin cancer. As such, excess miR-146a actually increases the activation of the NOTCH protein.
These results led Forloni et al. to test if delivering drugs that block the production of miR-146a by inhibiting skin cancer-causing genes, along with other drugs that inhibit the Notch signaling pathway, could be more effective than treatment with either drug on its own. The combined treatment was very effective against human skin cancer cells and could represent a promising development in the treatment of this disease.
DOI: http://dx.doi.org/10.7554/eLife.01460.002
doi:10.7554/eLife.01460
PMCID: PMC3927633  PMID: 24550252
miRNA; BRAF; NRAS; melanoma; human
14.  Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts 
Arthritis Research  1999;1(1):71-80.
Our aim was to identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with rheumatoid arthritis (RA). In RA, amplification of a distinct PCR product suitable for sequencing could be observed. Sequence analysis identified the PCR product as highly homologous to a 434 base pair segment of the human centromere kinesin-like protein CENP-E. Differential expression of CENP-E was confirmed by quantitative reverse transcription PCR, immunohistochemistry and in situ hybridization. CENP-E expression was independent from prednisolone and could not be completely inhibited by serum starvation. RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. Also, because motifs of CENP-E show homologies to jun and fos oncogene products and are involved in virus assembly, CENP-E may be involved in the pathophysiology of RA.
Introduction:
Articular destruction by invading synovial fibroblasts is a typical feature in rheumatoid arthritis (RA). Recent data support the hypothesis that key players in this scenario are transformed-appearing synovial fibroblasts at the site of invasion into articular cartilage and bone. They maintain their aggressive phenotype toward cartilage, even when first cultured and thereafter coimplanted together with normal human cartilage into severe combined immunodeficient mice for an extended period of time. However, little is known about the upregulation of genes that leads to this aggressive fibroblast phenotype. To inhibit this progressive growth without interfering with pathways of physiological matrix remodelling, identification of pathways that operate specifically in RA synovial fibroblasts is required. In order to achieve this goal, identification of genes showing upregulation restricted to RA synovial fibroblasts is essential.
Aims:
To identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with RA.
Methods:
RNA was extracted from cultured synovial fibroblasts from 10 patients with RA, four patients with osteoarthritis (OA), and one patient with psoriatic arthritis. RAP-PCR was performed using different arbitrary primers for first-strand and second-strand synthesis. First-strand and second-strand synthesis were performed using arbitrary primers: US6 (5' -GTGGTGACAG-3') for first strand, and Nuclear 1+ (5' -ACGAAGAAGAG-3'), OPN28 (5' -GCACCAGGGG-3'), Kinase A2+ (5' -GGTGCCTTTGG-3')and OPN24 (5' -AGGGGCACCA-3') for second-strand synthesis. PCR reactions were loaded onto 8 mol/l urea/6% polyacrylamide-sequencing gels and electrophoresed.Gel slices carrying the target fragment were then excised with a razor blade, eluated and reamplified. After verifying their correct size and purity on 4% agarose gels, the reamplified products derived from the single-strand confirmation polymorphism gel were cloned, and five clones per transcript were sequenced. Thereafter, a GenBank® analysis was performed. Quantitative reverse transcription PCR of the segments was performed using the PCR MIMIC® technique.In-situ expression of centromere kinesin-like protein-E (CENP-E) messenger (m)RNA in RA synovium was assessed using digoxigenin-labelled riboprobes, and CENP-E protein expression in fibroblasts and synovium was performed by immunogold-silver immunohistochemistry and cytochemistry. Functional analysis of CENP-E was done using different approaches (eg glucocorticoid stimulation, serum starvation and growth rate analysis of synovial fibroblasts that expressed CENP-E).
Results:
In RA, amplification of a distinct PCR product suitable for sequencing could be observed. The indicated complementary DNA fragment of 434 base pairs from RA mRNA corresponded to nucleotides 6615-7048 in the human centromere kinesin-like protein CENP-E mRNA (GenBank® accession No. emb/Z15005).The isolated sequence shared greater than 99% nucleic acid (P = 2.9e-169) identity with the human centromere kinesin-like protein CENP-E. Two base changes at positions 6624 (A to C) and 6739 (A to G) did not result in alteration in the amino acid sequence, and therefore 100% amino acid identity could be confirmed. The amplification of 10 clones of the cloned RAP product revealed the presence of CENP-E mRNA in every fibroblast culture examined, showing from 50% (271.000 ± 54.000 phosphor imager arbitrary units) up to fivefold (961.000 ± 145.000 phosphor imager arbitrary units) upregulation when compared with OA fibroblasts. Neither therapy with disease-modifying antirheumatic drugs such as methotrexate, gold, resochine or cyclosporine A, nor therapy with oral steroids influenced CENP-E expression in the RA fibroblasts. Of the eight RA fibroblast populations from RA patients who were receiving disease-modifying antirheumatic drugs, five showed CENP-E upregulation; and of the eight fibroblast populations from RA patients receiving steroids, four showed CENP-E upregulation.
Numerous synovial cells of the patients with RA showed a positive in situ signal for the isolated CENP-E gene segment, confirming CENP-E mRNA production in rheumatoid synovium, whereas in OA synovial tissue CENP-E mRNA could not be detected. In addition, CENP-E expression was independent from medication. This was further confirmed by analysis of the effect of prednisolone on CENP-E expression, which revealed no alteration in CENP-E mRNA after exposure to different (physiological) concentrations of prednisolone. Serum starvation also could not suppress CENP-E mRNA completely.
Discussion:
Since its introduction in 1992, numerous variants of the differential display method and continuous improvements including RAP-PCR have proved to have both efficiency and reliability in examination of differentially regulated genes. The results of the present study reveal that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts.
The mRNA, which has been found to be upregulated in rheumatoid synovial fibroblasts, codes for a kinesin-like motor protein named CENP-E, which was first characterized in 1991. It is a member of a family of centromere-associated proteins, of which six (CENP-A to CENP-F) are currently known. CENP-E itself is a kinetochore motor, which accumulates transiently at kinetochores in the G2 phase of the cell cycle before mitosis takes place, appears to modulate chromosome movement and spindle elongation,and is degraded at the end of mitosis. The presence or upregulation of CENP-E has never been associated with RA.
The three-dimensional structure of CENP-E includes a coiled-coil domain. This has important functions and shows links to known pathways in RA pathophysiology. Coiled-coil domains can also be found in jun and fos oncogene products, which are frequently upregulated in RA synovial fibroblasts. They are also involved in DNA binding and transactivation processes resembling the situation in AP-1 (Jun/Fos)-dependent DNA-binding in rheumatoid synovium. Most interestingly, these coiled-coil motifs are crucial for the assembly of viral proteins, and the upregulation of CENP-E might reflect the influence of infectious agents in RA synovium. We also performed experiments showing that serum starvation decreased, but did not completely inhibit CENP-E mRNA expression. This shows that CENP-E is related to, but does not completely depend on proliferation of these cells. In addition, we determined the growth rate of CENP-E high and low expressors, showing that it was independent from the amount of CENP-E expression. supporting the statement that upregulation of CENP-E reflects an activated RA fibroblast phenotype. In summary, the results of the present study support the hypothesis that CENP-E, presumably independently from medication, may not only be upregulated, but may also be involved in RA pathophysiology.
PMCID: PMC17776  PMID: 11056662
arthritis; centromere; differential display; immunohistochemistry; in situ hybridization; RNA fingerprinting
15.  microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma 
A moderate loss of miR-122 function correlates with up-regulation of seed-matched genes and down-regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti-miR-122 antagomir.Putative direct targets up-regulated with loss of miR-122 and secondary targets down-regulated with loss of miR-122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR-122 over- and under-expression.Loss of miR-122 secondary target expression in either tumorous or adjacent non-tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis-B or -C viruses (HBV or HCV) (But et al, 2008). Globally, the 5-year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)-expression profiles of tumor and adjacent non-tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR-122 was the most highly expressed miRNA in liver, and its expression was decreased almost two-fold in HCC tissue relative to adjacent non-tumor tissue, confirming earlier observations (Lagos-Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti-correlated with miR-122 expression. Consistent with the idea that transcripts anti-correlated with miR-122 are potential miR-122 targets, the most highly anti-correlated transcripts were highly enriched for the presence of the miR-122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell-cycle genes, the subset of seed-matched genes had no significant KEGG Pathway annotation, suggesting that miR-122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR-122 were not enriched for 3′UTR seed matches to miR-122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR-122 in vivo, silencing of miR-122 was performed by antisense inhibition (anti-miR-122) in wild-type mice (Figure 3). As with the genes negatively correlated with miR-122 in HCC patients, no significant biological annotation was associated with the seed-matched genes up-regulated by anti-miR-122 in mouse livers. The most significantly enriched biological annotation for anti-miR-122 down-regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down-regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR-122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti-correlation of proposed primary and secondary targets in non-healthy tissues.
To validate the direct correlation between miR-122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR-122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT-1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC-1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over-expressing miR-122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC-1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. To further validate the link of miR-122 and PGC-1α protein, we transfected PLC/PRF/5 cells with miR-122-expression vector, and observed an increase in PGC-1α protein levels. Importantly, transfection of both miR-122 mimetic and miR-122-expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti-miR-122 treatment increased lactate production. Together, the data support the function of miR-122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR-122-expression levels. However, miR-122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non-tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR-122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR-122 in liver physiology. We observed decreased expression of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 seemed to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC and to test whether increasing miR-122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
doi:10.1038/msb.2010.58
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival
16.  Increased activity and expression of histone deacetylase 1 in relation to tumor necrosis factor-alpha in synovial tissue of rheumatoid arthritis 
Arthritis Research & Therapy  2010;12(4):R133.
Introduction
The purpose of this study was to investigate the profile of histone deacetylase (HDAC) expression in the synovial tissue of rheumatoid arthritis (RA) compared with that of normal control and osteoarthritis (OA), and to examine whether there is a link between HDAC activity and synovial inflammation.
Methods
HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of total synovial tissue surgically obtained from normal, OA and RA joints. The level of cytoplasmic tumor necrosis factor a (TNFα) fraction was measured by ELISA. Total RNA of synovial tissue was used for RT-PCR of HDAC1-8. In synovial fibroblasts from RA (RASFs), the effects of TNFα on nuclear HDAC activity and class I HDACs (1, 2, 3, 8) mRNA expressions were examined by quantitative real-time PCR. The protein expression and distribution of class I HDACs were examined by Western blotting.
Results
Nuclear HDAC activity was significantly higher in RA than in OA and normal controls and correlated with the amount of cytoplasmic TNFα. The mRNA expression of HDAC1 in RA synovial tissue was higher than in OA and normal controls, and showed positive correlation with TNFα mRNA expression. The protein level of nuclear HDAC1 was higher in RA synovial tissue compared with OA synovial tissue. Stimulation with TNFα significantly increased the nuclear HDAC activity and HDAC1 mRNA expression at 24 hours and HDAC1 protein expression at 48 hours in RASFs.
Conclusions
Our results showed nuclear HDAC activity and expression of HDAC1 were significantly higher in RA than in OA synovial tissues, and they were upregulated by TNFα stimulation in RASFs. These data might provide important clues for the development of specific small molecule HDAC inhibitors.
doi:10.1186/ar3071
PMCID: PMC2945023  PMID: 20609223
17.  Comprehensive microRNA Profiling of Prostate Cancer 
Journal of Cancer  2013;4(5):350-357.
MicroRNAs are small non-coding RNA molecules that have been shown to regulate the expression of genes linked to cancer. The relevance of microRNAs in the development, progression and prognosis of prostate cancer is not fully understood. It is also possible that these specific molecules may assist in the recognition of aggressive tumors and the development of new molecular targets. Our study investigated the importance of several microRNAs in cases of prostate cancer from 37 patients that were manually microdissected to obtain pure populations of tumor cells, normal epithelium and adjacent stroma. MicroRNA was extracted for PCR array profiling. Differentially expressed miRNAs for each case were used to compare tumor vs. normal epithelium and tumor-adjacent stroma samples.
Loss of 18 miRNAs (e.g.miR-34c, miR-29b, miR-212 and miR-10b) and upregulation of miR-143 and miR-146b were significantly found in all the tumors in comparison with normal epithelium and/or stroma (p≤ 0.001). A different signature was found in the high grade tumors (Gleason score ≥ 8) when compared with tumors Gleason score 6. Upregulation of miR-122, miR-335, miR-184, miR-193, miR-34, miR-138, miR-373, miR-9, miR-198, miR-144 and miR-215 and downregulation of miR-96, miR-222, miR-148, miR-92, miR-27, miR-125, miR-126, miR-27 were found in the high grade tumors.
MicroRNA profiling in prostate cancer appears to have unique expression patterns in comparison with normal tissue. These differential expressed miRNAs may provide novel diagnostic and prognostic tools that will assist in the recognition of prostate cancers with aggressive behavior.
doi:10.7150/jca.6394
PMCID: PMC3677622  PMID: 23781281
microRNA; Prostate Cancer; biomarkers.
18.  miR-146a–Mediated Extracellular Matrix Protein Production in Chronic Diabetes Complications 
Diabetes  2011;60(11):2975-2984.
OBJECTIVE
microRNAs (miRNAs), through transcriptional regulation, modulate several cellular processes. In diabetes, increased extracellular matrix protein fibronectin (FN) production is known to occur through histone acetylator p300. Here, we investigated the role of miR-146a, an FN-targeting miRNA, on FN production in diabetes and its relationship with p300.
RESEARCH DESIGN AND METHODS
miR-146a expressions were measured in endothelial cells from large vessels and retinal microvessels in various glucose levels. FN messenger RNA expression and protein levels with or without miR-146a mimic or antagomir transfection were examined. A luciferase assay was performed to detect miR-146a’s binding to FN 3′–untranslated region (UTR). Likewise, retinas from type 1 diabetic rats were studied with or without an intravitreal injection of miR-146a mimic. In situ hybridization was used to localize retinal miR-146a. Cardiac and renal tissues were analyzed from type 1 and type 2 diabetic animals.
RESULTS
A total of 25 mmol/L glucose decreased miR-146a expression and increased FN expression compared with 5 mmol/L glucose in both cell types. miR-146a mimic transfection prevented such change, whereas miR-146a antagomir transfection in the cells in 5 mmol/L glucose caused FN upregulation. A luciferase assay confirmed miR-146a’s binding to FN 3′-UTR. miR-146a was localized in the retinal endothelial cells and was decreased in diabetes. Intravitreal miR-146a mimic injection restored retinal miR-146a and decreased FN in diabetes. Additional experiments showed that p300 regulates miR-146a. Similar changes were seen in the retinas, kidneys, and hearts in type 1 and type 2 diabetic animals.
CONCLUSIONS
These studies showed a novel, glucose-induced molecular mechanism in which miR-146a participates in the transcriptional circuitry regulating extracellular matrix protein production in diabetes.
doi:10.2337/db11-0478
PMCID: PMC3198068  PMID: 21885871
19.  Involvement of inflammation-related miR-155 and miR-146a in diabetic nephropathy: implications for glomerular endothelial injury 
BMC Nephrology  2014;15:142.
Background
MicroRNAs have been demonstrated to play an important role in the pathogenesis of diabetic nephropathy (DN). In this study, we investigated both the repertoire of miRNAs in the kidneys of patients with DN and their potential regulatory role in inflammation-mediated glomerular endothelial injury.
Methods
The miRNA expression profiling of the renal biopsy samples was performed by a microarray analysis; then, in situ hybridization and real-time polymerase chain reaction (PCR) were used to determine the localization and expression of two of the miRNAs significantly up-regulated in human DN kidney samples, miR-155 and miR-146a, in the kidney tissues from type 1 and type 2 DN rat models. Human renal glomerular endothelial cells (HRGECs) cultured under high-glucose conditions were transfected with miR-155 and miR-146a mimics, and the transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, and nuclear factor (NF)-κB expressions were examined by western blot, real-time PCR, and an electrophoresis mobility shift assay.
Results
The expression of both miR-155 and miR-146a was increased more than fivefold in the kidney samples of the DN patients compared with the controls, and the miR-155 expression was closely correlated with the serum creatinine levels (R = 0.95, P = 0.004). During the induction and progression of the disease in type 1 and type 2 DN rat models, miR-155 and miR-146a were demonstrated to increase gradually. In vitro, high glucose induced the over-expression of miR-155 and miR-146a in the HRGECs, which, in turn, increased the TNF-α, TGF-β1, and NF-κB expression.
Conclusions
Taken together, these findings indicate that the increased expression of miR-155 and miR-146a in the DN patients and in the experimental DN animal models was found to contribute to inflammation-mediated glomerular endothelial injury.
doi:10.1186/1471-2369-15-142
PMCID: PMC4236663  PMID: 25182190
20.  Activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor PTEN at sites of invasive growth and destruction 
Arthritis Research  1999;2(1):59-64.
In the present study, we searched for mutant PTEN transcripts in aggressive rheumatoid arthritis synovial fibroblasts (RA-SF) and studied the expression of PTEN in RA. By automated sequencing, no evidence for the presence of mutant PTEN transcripts was found. However, in situ hybridization on RA synovium revealed a distinct expression pattern of PTEN, with negligible staining in the lining layer but abundant expression in the sublining. Normal synovial tissue exhibited homogeneous staining for PTEN. In cultured RA-SF, only 40% expressed PTEN. Co-implantation of RA-SF and normal human cartilage into severe combined immunodeficiency (SCID) mice showed only limited expression of PTEN, with no staining in those cells aggressively invading the cartilage. Although PTEN is not genetically altered in RA, these findings suggest that a lack of PTEN expression may constitute a characteristic feature of activated RA-SF in the lining, and may thereby contribute to the invasive behaviour of RA-SF by maintaining their aggressive phenotype at sites of cartilage destruction.
Aims:
PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin. Mutations of PTEN have been described in several human cancers and associated with their invasiveness and metastatic properties. Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth. In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF. In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA.
Methods:
Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals. Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far. The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing. For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription. For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared. To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice. After 60 days, mice were sacrificed, the implants removed and embedded into paraffin.
Results:
PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig. 1). No additional bands that could account for mutant PTEN variants were detected. Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051). However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes. As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA. In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b). Normal synovial specimens showed homogeneous staining for PTEN within the thin synovial membrane (Fig. 2c). In situ hybridization using the sense probe gave no specific staining (Fig. 2d). We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage. Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig. 3a), and the proportion of PTEN expressing cells did not change throughout the passages. In contrast, control experiments using a specific RNA probe for MMP-2 revealed mRNA expression by nearly all cultured cells (Fig. 3b). As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation. Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig. 3c). In situ hybridization for MMP-2 showed abundant staining in these cells (Fig. 3d).
Discussion:
Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more than half of cultured RA-SF is of interest. It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively.
It has been proposed that the tyrosine phosphatase activity of PTEN is responsible for its tumour suppressor activity by counteracting the actions of protein tyrosine kinases. As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease. However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling. The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-β. Therefore, it could be hypothesized that TGF-β might be responsible for the downregulation of PTEN. However, the expression of TGF-β is not restricted to the lining but found throughout the synovial tissue in RA. Moreover, in our study the percentage of PTEN expressing RA-SF remained stable for six passages in culture, whereas molecules that are cytokine-regulated in vivo frequently change their expression levels when cultured over several passages. Also, cultured RA-SF that were implanted into SCID mice and deeply invaded the cartilage did not show significant expression of PTEN after 60 days. The drop in the percentage of PTEN expressing cells from the original cell cultures to the SCID mouse implants is of interest as this observation goes along with data from previous studies that have shown the prominent expression of activation-related molecules in the SCID mice implants that in vivo are found predominantly in the lining layer. Therefore, our data point to endogenous mechanisms rather than to the influence of exogenous human cytokines or factors in the downregulation of PTEN. Low expression of PTEN may belong to the features that distinguish between the activated phenotype of RA-SF and the sublining, proliferating but nondestructive cells.
PMCID: PMC17804  PMID: 11219390
rheumatoid arthritis; synovial membrane; fibroblasts; PTEN tumour suppressor; severe combined immunodeficiency (SCID) mouse model; cartilage destruction; in situ hybridization
21.  MicroRNA expression in Epstein-Barr virus-associated post-transplant smooth muscle tumours is related to leiomyomatous phenotype 
Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT) are rare complications. In our previous molecular analysis, we have evaluated the expression of regulatory microRNA which are known to be EBV-related (miR-146a and miR-155) but found no deregulation in PTSMT. In this current analysis, we aimed to characterize the expression profiles of several hundred microRNA. Tissue samples from PTSMT and uterine leiomyomas were analysed by quantitative real-time PCR for the expression of 365 mature microRNA. PTSMT and leiomyomas share a highly similar microRNA profile, e.g. strong expression of miR-143/miR-145 cluster and low expression of miR-200c. Among EBV-related microRNA (miR-10b, miR-21, miR-29b, miR-34a, miR-127, miR-146a, miR-155, miR-200b, miR-203 and miR-429) only miR-10b and miR-203 were significantly deregulated. The expression pattern of microRNA in PTSMT is not associated with EBV infection but reflects the leiomyomatous differentiation of the tumour cells.
doi:10.1186/2045-3329-3-9
PMCID: PMC3706214  PMID: 23830214
Epstein-Barr virus; EBV; Post-transplant smooth muscle tumour; PTSMT; MicroRNA
22.  microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer 
Molecular Cancer  2012;11:71.
Background
Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles.
Results
The expression of miR-146a was evaluated by quantitative PCR (qPCR) and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10) and COP9 signalosome complex subunit 8 (COPS8), as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR) pathway of nuclear factor-kappaB (NF-kappaB) activation. Lysophosphatidic acid (LPA) induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction.
Conclusions
miR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in the regulation of NF-kappaB activity.
doi:10.1186/1476-4598-11-71
PMCID: PMC3515505  PMID: 22992343
Stomach cancer; Non-coding RNA; Cytokines
23.  MicroRNAs Expression in Triple Negative vs Non Triple Negative Breast Cancer in Tunisia: Interaction with Clinical Outcome 
PLoS ONE  2014;9(11):e111877.
Introduction
MicroRNAs are small, non coding regulatory molecules containing approximately 21 to 25 nucleotides. They function as controllers of expression at post transcriptional levels of most human protein-coding genes and play an essential role in cell signaling pathways. The objective of the present study is to evaluate the expression profile of the following micro-RNAs: miR-10b, miR-17, miR-21, miR-34a, miR-146a, miR-148a and miR-182, and to determine their possible interaction in triple-negative and non triple-negative primary breast cancers based on clinical outcome.
Methods
60 triple-negative and non triple-negative breast cancer cases, along with their corresponding normal samples were investigated in relation to the expression of the seven studied miRNAs using qPCR Syber Green.
Results
We observed that miR-21, miR-146a and miR-182 were significantly over expressed in triple negative breast cancer. Moreover, miR-10b, miR-21 and miR-182 were significantly associated to lymph node metastases occurrence in triple negative breast carcinoma while only miR-10b was associated with grade III in non triple negative breast cancer cases. Almost all the analyzed microRNAs were strongly associated with patients’ genico-obstetric history in non triple negative breast cancer cases except for miR-34a. All the studied microRNAs were strongly correlated with the use of the contraceptive pills in non triple negative breast cancer groups. The additive effect of hormonal factors in triple negative breast cancer cases showed an association with all the studied miRs except for miR-34 and miR-146a.
Conclusion
The studied microRNAs are strongly influenced by environmental factors especially with hormonal patients’ history. Moreover, miR-10b, miR-21 and miR-182 could be defined as biomarkers in breast cancer to predict both lymph node metastases and grade III occurrence.
doi:10.1371/journal.pone.0111877
PMCID: PMC4219794  PMID: 25369070
24.  Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection 
Background
Hepatitis C Virus (HCV), a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA) regulate inflammation (miR-155, -146a and -125b) as well as hepatocyte function (miR-122).
Methods
Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection.
Results
We found that monocytes from chronic HCV infected treatment-naïve (cHCV) but not treatment responder patients showed increased expression of miR-155, a positive regulator of TNFα, and had increased TNFα production compared to monocytes of normal controls. After LPS stimulation, miR-155 levels were higher in monocytes from cHCV patients compared to controls. MiR-125b, which has negative regulatory effects on inflammation, was decreased in cHCV monocytes compared to controls. Stimulation of normal monocytes with TLR4 and TLR8 ligands or HCV core, NS3 and NS5 recombinant proteins induced a robust increase in both miR-155 expression and TNFα production identifying potential mechanisms for in vivo induction of miR-155. Furthermore, we found increased serum miR-155 levels in HCV patients compared to controls. Serum miR-125b and miR-146a levels were also increased in HCV patients. Serum levels of miR-122 were elevated in cHCV patients and correlated with increased ALT and AST levels and serum miR-155 levels.
Conclusion
In conclusion, our novel data demonstrate that miR-155, a positive regulator of inflammation, is upregulated both in monocytes and in the serum of patients with chronic HCV infection. Our study suggests that HCV core, NS3, and NS5 proteins or TLR4 and TLR8 ligands can mediate increased miR-155 and TNFα production in chronic HCV infection. The positive correlation between serum miR-155 and miR-122 increase in cHCV may be an indicator of inflammation-induced hepatocyte damage.
doi:10.1186/1479-5876-10-151
PMCID: PMC3477071  PMID: 22846613
Biomarker; MiR-122; MiR-155; MiR-125b; ALT; Inflammation
25.  miR-21 and miR-375 microRNAs as candidate diagnostic biomarkers in squamous cell carcinoma of the larynx: association with patient survival 
The dismal outcome of laryngeal squamous cell carcinoma (SCC) patients highlights the need for novel prognostic biomarkers. The involvement of microRNAs in cancer and their potential as biomarkers of diagnosis and prognosis are becoming increasingly appreciated. We sought to identify microRNAs that exhibit altered expression in laryngeal SCC and to determine whether microRNA (miRNA) expression is predictive of disease progression and/or patient survival. The expression of two miRNAs, miR-21 and miR-375, was evaluated using total RNA isolated from freshly-frozen primary tumors and non-cancerous laryngeal squamous epithelial tissues and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. We further analyzed the association between the expression of miRNAs and the clinicopathological features. A marked difference in the microRNA expression pattern was observed between tumors and non-cancerous tissue. MiR-21 and miR-375 were expressed at higher and lower levels, respectively, in the laryngeal SCC samples, compared to the normal samples (p < 0.01 and p < 0.001, respectively). There was no correlation between characteristics such as age, sex, clinical stage, and alcohol use, and the expression level of mir-21. The relative expression of mir-375 in laryngeal SCC was shown to be associated with localization of the tumor in these patients (p = 0.037) and with alcohol use (p < 0.05). Patients with high miR-21 or low miR-375 expression in tumor tissues had poorer prognoses compared to patients with lower miR-21 or higher miR-375 expression. Furthermore, the miR-21/miR-375 expression ratio was highly sensitive (0.94) and specific (0.94) for disease prediction. These data suggest that the pattern of microRNA expression in primary laryngeal SCC tissues is reflective of the disease status and that miR-21 and miR-375 expression levels, in particular, may serve as potential biomarkers with applications in the clinical setting.
PMCID: PMC4212934  PMID: 25360224
Laryngeal squamous cell carcinoma; microRNA; biomarker; prognosis

Results 1-25 (1311542)