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      miRNA-221-3p Enhances the Secretion of Interleukin-4 in Mast Cells through the Phosphatase and Tensin Homolog/p38/Nuclear Factor-kappaB Pathway

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          Abstract

          Mast cells play a central role in asthma. Moreover, serum miRNA-221-3p ( miR-221) has been shown to be markedly increased in children with asthma. In the current study, we aimed to examine miR-221 expression in an asthma model and elucidate the mechanisms regulating interleukin (IL)-4 secretion in mast cells. Using polymerase chain reaction, we found that miR-221 was upregulated in a murine asthma model and in P815 mast cells after lipopolysaccharide (LPS) stimulation. Moreover, miR-221 upregulated IL-4 secretion from P815 cells, as shown by enzyme-linked immunosorbent assays. Bioinformatics analysis, luciferase reporter gene assays, and western blotting showed that phosphatase and tensin homolog (PTEN) was a target of miR-221 and could block IL-4 secretion stimulated by miR-221. The phosphorylation of p38 (protein) and activity of nuclear factor-kappaB (NF-κB) were increased after overexpression of miR-221, as shown by electrophoretic mobility shift assays. Finally, treatment with specific inhibitors could block IL-4 secretion. In conclusion, miR-221, which was overexpressed in a murine asthma model, stimulated IL-4 secretion in mast cells through a pathway involving PTEN, p38, and NF-κB.

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          The role of the mast cell in the pathophysiology of asthma.

          There is compelling evidence that human mast cells contribute to the pathophysiology of asthma. Mast cells, but not T cells or eosinophils, localize within the bronchial smooth muscle bundles in patients with asthma but not in normal subjects or those with eosinophilic bronchitis, a factor likely to be important in determining the asthmatic phenotype. The mechanism of mast cell recruitment by asthmatic airway smooth muscle involves the CXCL10/CXCR3 axis, and several mast cell mediators have profound effects on airway smooth muscle function. The autacoids are established as potent bronchoconstrictors, whereas the proteases tryptase and chymase are being demonstrated to have a range of actions consistent with key roles in inflammation, tissue remodeling, and bronchial hyperresponsiveness. IL-4 and IL-13, known mast cell products, also induce bronchial hyperresponsiveness in the mouse independent of the inflammatory response and enhance the magnitude of agonist-induced intracellular Ca2+ responses in cultured human airway smooth muscle. There are therefore many pathways by which the close approximation of mast cells with airway smooth muscle cells might lead to disordered airway smooth muscle function. Mast cells also infiltrate the airway mucous glands in subjects with asthma, showing features of degranulation, and a positive correlation with the degree of mucus obstructing the airway lumen, suggesting that mast cells play an important role in regulating mucous gland secretion. The development of potent and specific inhibitors of mast cell secretion, which remain active when administered long-term to asthmatic airways, should offer a novel approach to the treatment of asthma.
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            Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues.

            Global micro-RNA (miR) profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used frozen tissues. However, formalin fixation is the standard and routine histological practice for optimal preservation of cellular morphology. To determine whether miR analysis of formalin-fixed tissues is feasible, quantitative real-time PCR (qRT-PCR) profiling of miR expression in 40 archival formalin-fixed paraffin-embedded (FFPE) breast lumpectomy specimens were performed. Taqman Low Density Arrays (TLDAs) were used to assess the expression level of 365 miRs in 34 invasive ductal carcinomas and in 6 normal comparators derived from reduction mammoplasties. Its technical reproducibility was high, with intra-sample correlations above 0.9 and with 92.8% accuracy in differential expression comparisons, indicating such global profiling studies to be technically and biologically robust. The TLDA data were confirmed using conventional single-well qRT-PCR analysis, showing a strong and statistically significant concordance between these two methods. Paired frozen and FFPE breast cancer samples from the same patients showed a similar level of robust correlation of at least 0.94. Compared with normal breast samples, a panel of miRs was consistently dysregulated in breast cancer, including earlier-reported breast cancer-related miRs, such as upregulated miR-21, miR-155, miR-191, and miR-196a, and downregulated miR-125b and miR-221. Additional novel miR sequences of potential biological relevance were also uncovered. These results show the validity and utility of conducting global miR profiling using FFPE samples, thereby offering enormous opportunities to evaluate archival banks of such materials, linked to clinical databases, to rapidly acquire greater insight into the clinically relevant role for miRs in human malignancies.
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              Targeting TNF-alpha: a novel therapeutic approach for asthma.

              Approximately 5% to 10% of patients with asthma have severe disease that is refractory or poorly responsive to inhaled corticosteroid therapy. These patients represent an important unmet clinical need because they experience considerable morbidity and mortality and consume a disproportionately large amount of health care resources. TNF-alpha is a proinflammatory cytokine that has been implicated in many aspects of the airway pathology in asthma. Evidence is emerging to suggest that it might play an important role in severe refractory disease. The development of novel TNF-alpha antagonists has allowed us to test the role of this cytokine in vivo. Preliminary studies have demonstrated an improvement in asthma quality of life, lung function, and airway hyperresponsiveness and a reduction in exacerbation frequency in patients treated with anti-TNF-alpha therapy. However, there is marked heterogeneity in response, suggesting that benefit is likely to be reserved to a small subgroup. Importantly, where efficacy is reported, this also needs to be considered in the context of concerns about the safety of anti-TNF-alpha therapies. Therefore the challenge for clinicians is to evaluate the risk/benefit ratio of these therapies in individual patients with asthma.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 February 2016
                2016
                : 11
                : 2
                : e0148821
                Affiliations
                [1 ]Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu, China 210008
                [2 ]Department of Pediatrics, Jiangsu Huai'an Maternity and Children's Hospital, Huai'an, Jiangsu, China 223002
                National University of Singapore, SINGAPORE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: DZ FL. Performed the experiments: YZ QY HX JZ HD HG JY. Analyzed the data: YZ QY HX. Contributed reagents/materials/analysis tools: YZ QY HX JZ HD. Wrote the paper: YZ QY.

                Article
                PONE-D-15-36939
                10.1371/journal.pone.0148821
                4764704
                26901347
                c821f4e9-4110-4c71-9269-8b5914007ddb
                © 2016 Zhou et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 September 2015
                : 21 January 2016
                Page count
                Figures: 7, Tables: 1, Pages: 14
                Funding
                The current research was supported by the National Natural Science Foundation of China (81200012, Feng Liu; 81370132, Deyu Zhao; http://www.nsfc.gov.cn/), by Nanjing Medical Science and Technique Development Foundation ( http://se.njh.gov.cn/index/index.do) and by Nanjing Public Health Bureau (ZKX11012, Deyu Zhao; http://www.njh.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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