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Negative/null result

The expression profile of miR-23b is not altered in peripheral blood mononuclear cells of patients with idiopathic inflammatory myopathies

[version 1; peer review: 2 approved]
+ Deceased author
PUBLISHED 23 Oct 2013
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Abstract

Idiopathic inflammatory myopathies (IIM) belong to a group of autoimmune disorders, primarily characterized by chronic inflammation of human skeletal muscle tissue. The etiology of these diseases is unknown, however, genetic predisposition plays a significant role in disease onset. Beside the known genetic risk located in the MHC complex, the epigenetic modifications including changes in miRNAs expression profiles have been recently implicated recently in many autoimmune diseases. Micro RNA molecules are involved in many physiological processes, including the regulation of the immune response.
In our study we have focused on the miR-23b, as it represents a novel promising autoimmunity regulator molecule. Downregulation of miR-23b was recently described in patients with rheumatoid arthritis and systemic lupus erythematosus. We have measured the expression miR-23b peripheral blood mononuclear cells of patients with dermatomyositis and polymyositis. No meaningful difference was found in comparison with healthy controls.

Introduction and aims

Idiopathic inflammatory myopathies (IIM) are chronic autoimmune disorders characterized by skeletal muscle weakness, inflammatory immune cells in muscle tissues and frequent presence of serum autoantibodies. IIM can be divided into several groups: dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), and immune mediated necrotizing myopathy (NM). The etiology of IIM remains unclear; however current findings indicate that epigenetic mechanisms may contribute to the disease pathogenesis1,2.

MicroRNA molecules are part of the epigenetic regulatory network. They act as negative regulators of gene expression and participate in the regulation of key biological processes such as cell growth, differentiation, proliferation or apoptosis36. Recent findings highlight the crucial importance of miRNAs in development, homeostasis and function of innate and adaptive immunity710. Aberrant expression patterns have been documented in a broad range of diseases including autoimmune disorders11.

In our study we have focused on the miR-23b type of microRNA, as it represents a novel promising autoimmunity regulator molecule with considerable therapeutic potential12. Recent research has revealed downregulation of this miRNA in resident cells present in inflammatory lesions of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and its role during the pathogenesis of autoimmune disease has been functionally characterized12.

The aim of our study was to analyze differential expression of miR-23b and miR-23b* in peripheral blood mononuclear cells (PBMCs) of patients with PM/DM, and to examine potential involvement of these miRNA types in the pathogenesis of IIM.

Material and methods

A cohort of 25 adult patients (age range 21–86 years, 7 males) suffering from idiopathic inflammatory myopathy and 22 healthy controls (age range 25–79 years, 5 males), both from the middle region of Czech Republic were analyzed. Diagnosis of PM or DM was determined according to the Bohan and Peter criteria13,14. In total 12 of patients suffered from PM, 13 patients were diagnosed as DM. All individuals involved in this study signed an informed consent. PBMCs of all individuals were obtained and purified by density gradient centrifugation on Ficoll in Leucosep tubes (Greiner Bio-One GmbH, Germany). 10 ml of peripheral blood of patients with myositis and healthy control subjects was collected into EDTA coated tubes under sterile conditions. The blood was added to a LeucoSep tube. The cell separation tubes were centrifuged (Jouan CR 3i; Jouan, St. Herblain, France) for 10 min at 2400 rpm without braking at room temperature. The cell suspension was collected, and the cells were washed three times in PBS (for 10 min at 1000 rpm), resuspended in 1 ml PBS and transferred into a 1,5 ml microcentrifuge tube. The microcentrifuge tube was centrifuged at 8000 rpm for 1 minute, supernatant was removed and the cell pellet was frozen using liquid nitrogen and stored at -80°C. The total RNA preparation from PBMCs was carried out using the conventional TRIZOL® reagent (Invitrogen, Carlsbad, USA) extraction procedure15. Afterwards, the RNA was treated with RQ1 RNase free DNase (Promega, USA) for 20 min at 37°C in order to remove genomic DNA contamination, purified with phenol-chloroform-isoamylalcohol (25:24:1, PENTA, Czech Republic) reextraction and precipitated by 1 volume 2-propanol and 1/10 volume 300 mM sodium acetate (pH 4.8). Then, the RNA was washed twice with 70% ethanol and dissolved in RNase-free water (Life Technologies, Carlsbad, CA, USA). The quantity of total RNA was measured with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, USA). A microfluidic electrophoresis 2100 Bioanalyzer (Agilent Technologies, USA) was then used to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%].

TaqMan gene specific miRNA assays were used to quantify the expression levels of mature miR-23b and miR-23b* (microRNA 000400, 002126) from Life Technologies (Carlsbad, CA, USA). Total RNA was reverse-transcribed by the TaqMan microRNA reverse transcription kit (Applied Biosystems) in a reaction mixture containing a miR-specific stem-loop reverse transcription (RT) primer. The quantification of mature miRNAs was performed using the TaqMan miRNA assay kit (Applied Biosystems) containing TaqMan primers in a universal PCR master mix without AmpErase UNG. 4 small nuclear RNAs - RNU44, RNU48, U47 and RNU6B (microRNA 001094, 001006, 001223, 001093, Applied Biosystems) were amplified as an internal control. qPCR was conducted at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression level of miR-23b (miR-23b*) is shown as delta Ct. The delta Ct value was normalized to the mean value of four other miRNA molecules used in this experiment as housekeeping molecules (RNU44, RNU48, RNU6B, U47). This process was used to overcome the differences in cDNA quantity between different samples. All analyses were done in technical duplicates.

Statistical significance was determined by the Student's t-test using Microsoft Excel®2007. A value of P<0.05 was considered to be statistically significant.

The whole study, including the laboratory work was performed at the Institute of Rheumatology in Prague. Collection of clinical data and blood from patients as well as the laboratory analysis of the biological material was undertaken under the regulation of the relevant local research ethics committee (Ref. Nr. 5371/2012).

Results and discussion

We have examined the differential expression of the miR-23b and miR-23b* in PBMCs of patients with PM/DM and healthy controls. The detection of expression of miR-23b and miR-23b* was performed by quantitative real-time PCR. The median expression level (delta Ct) of miR-23b in the group of IIM patients was -3.90, the expression (delta Ct) in the control group reached -4.03. The median expression level of miR-23b* was -11.69 in IIM patients and -11.68 in the controls (Figure 1). The expression level (delta Ct) of both tested miR-23b molecules did not show any significant differences between patients suffering from myositis and control cohort (Figure 1). Furthermore, we did not find any difference in miR-23b (or miR-23b*) expression between DM and PM patients. The table with raw qPCR expression data used for statistical evaluation is provided in supplementary material.

35f27e54-2510-498e-b6d5-1e6ed9a1de95_figure1.gif

Figure 1. Regulation of the miR-23b and miR-23b* expression in patients and controls.

The expression level of miR-23b was not significantly changed in patients suffering from idiopathic inflammatory myopathy when compared with healthy controls (A). No significant difference in expression of miR-23b* was found between myositis patients and controls (B).

The role of miR-23b has been studied on inflammatory lesions of humans with lupus erythematosus or rheumatoid arthritis, as well as on corresponding tissues of murine models of lupus, rheumatoid arthritis and multiple sclerosis12. The authors Zhu et al.12 demonstrated that miR-23b targets TAB2, TAB3 and IKK-α mRNA transcripts and thereby suppresses autoimmune symptomatology by limiting the actions of the key proinflammatory cytokines IL-17, IL-1β and TNF. As it was found that the in vitro treatment of resident cell lines with IL-17 led to the downregulation of miR-23b, the expression of miR-23b and IL-17 in tissue resident cells seems to be mutually antagonistic12. IL-17 is mainly produced by activated T-cells, has the ability to activate other T-cells, and is also capable of triggering the inflammatory process in many other tissues/cells16,17. Involvement of miR-23b in the pathogenesis of myositic muscle was recently reported12 and the etiopathogenetic role of IL-17 in IIM has also been previously described18,19. The aim of our research was to analyze the changes in expression of miR-23b/miR-23b* in PBMCs from patients with myositis and to evaluate the importance of the IL-17/miR-23b signaling network in the PMBCs of these patients. Our results clearly show that the expression profile of miR-23b (as well as miR-23b*) is not changed in the PBMCs of IIM patients. Therefore, the regulative role of miR-23b in the development of myositis is not taking place in PMBCs. We cannot exclude involvement of this system in the local inflammatory process within the inflamed myositis muscle tissue.

Comments on this article Comments (1)

Version 1
VERSION 1 PUBLISHED 23 Oct 2013
  • Reader Comment 29 Oct 2013
    Paul Dominguez-Gutierrez, University of Florida, USA
    29 Oct 2013
    Reader Comment
    This is potentially a very interesting paper if miR-23b is selective for certain autoimmune diseases over others. There are several things that could greatly improve the data however.

    1) Report the ... Continue reading
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Remakova M, Svitalkova T, Skoda M et al. The expression profile of miR-23b is not altered in peripheral blood mononuclear cells of patients with idiopathic inflammatory myopathies [version 1; peer review: 2 approved]. F1000Research 2013, 2:223 (https://doi.org/10.12688/f1000research.2-223.v1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 23 Oct 2013
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Reviewer Report 11 Dec 2013
Stefano Casola, Genetics and Epigenetics of Lymphocyte Development and Transformation Research Program, Institute of Molecular Oncology Foundation (IFOM Institute), Milan, Italy 
Approved
VIEWS 16
In this article the authors provide convincing evidence that expression of miRNas mir23 and mir23b* in peripheral blood mononuclear cells of patients affected by inflammatory myopathies is unchanged. This result warrants future investigation to elucidate the role of the same ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Casola S. Reviewer Report For: The expression profile of miR-23b is not altered in peripheral blood mononuclear cells of patients with idiopathic inflammatory myopathies [version 1; peer review: 2 approved]. F1000Research 2013, 2:223 (https://doi.org/10.5256/f1000research.2627.r2169)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 11 Nov 2013
Maria Rescigno, Department of Experimental Oncology, European Institute of Oncology, Milan, Italy 
Approved
VIEWS 17
Overall I approve of this article; I would just add in the discussion that one caveat of this work is the use of PBMC and that by taking resident inflammatory cells the authors may be able to see a difference ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Rescigno M. Reviewer Report For: The expression profile of miR-23b is not altered in peripheral blood mononuclear cells of patients with idiopathic inflammatory myopathies [version 1; peer review: 2 approved]. F1000Research 2013, 2:223 (https://doi.org/10.5256/f1000research.2627.r2167)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

Comments on this article Comments (1)

Version 1
VERSION 1 PUBLISHED 23 Oct 2013
  • Reader Comment 29 Oct 2013
    Paul Dominguez-Gutierrez, University of Florida, USA
    29 Oct 2013
    Reader Comment
    This is potentially a very interesting paper if miR-23b is selective for certain autoimmune diseases over others. There are several things that could greatly improve the data however.

    1) Report the ... Continue reading
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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