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      Identification of miRNA Reference Genes in Extracellular Vesicles from Adipose Derived Mesenchymal Stem Cells for Studying Osteoarthritis

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          Abstract

          Osteoarthritis (OA) leads to chronic pain and disability, and traditional conservative treatments are not effective in the long term. The intra-articular injection of mesenchymal stem cells (MSCs) is considered a novel therapy for OA whose efficacy mainly relies on the adaptive release of paracrine molecules which are either soluble or extracellular vesicles (EVs) embedded. The correct quantification of EV-miRNAs using reliable reference genes (RGs) is a crucial step in optimizing this future therapeutic cell-free approach. The purpose of this study is to rate the stabilities of literature-selected proposed RGs for EV-miRNAs in adipose derived-MSCs (ASCs). EVs were isolated by ultracentrifugation from ASCs cultured with or without inflammatory priming mimicking OA synovial fluid condition. Expression of putative RGs (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA) was scored by using the algorithms geNorm, NormFinder, BestKeeper and ΔCt method. miR-16a-5p/miR-23a-3p yielded the most stable RGs, whereas let-7a-5p/miR-425-5p performed poorly. Outcomes were validated by qRT-PCR on miR-146a-5p, reported to be ASC-EVs enriched and involved in OA. Incorrect RG selection affected the evaluation of miR-146a-5p abundance and modulation by inflammation, with both values resulting strongly donor-dependent. Our findings demonstrated that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for ASC-EVs miRNAs evaluation. A correct approach would increase the accuracy of embedded molecule assessments aimed to develop therapeutic strategies for the treatment of OA based on EVs.

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          Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

          Background Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Results Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Conclusion Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.
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            MiRNA-Mediated Macrophage Polarization and its Potential Role in the Regulation of Inflammatory Response.

            Monocytes and macrophages are important components of the immune system, specialized in either removing pathogens as part of innate immunity or contributing to adaptive immunity through antigen presentation. Essential to such functions is classical activation (M1) and alternative activation (M2) of macrophages. M1 polarization of macrophages is characterized by production of pro-inflammatory cytokines, antimicrobial and tumoricidal activity, whereas M2 polarization of macrophages is linked to immunosuppression, tumorigenesis, wound repair, and elimination of parasites. MiRNAs are small non-coding RNAs with the ability to regulate gene expression and network of cellular processes. A number of studies have determined miRNA expression profiles in M1 and M2 polarized human and murine macrophages using microarray and RT-qPCR arrays techniques. More specifically, miR-9, miR-127, miR-155, and miR-125b have been shown to promote M1 polarization while miR-124, miR-223, miR-34a, let-7c, miR-132, miR-146a, and miR-125a-5p induce M2 polarization in macrophages by targeting various transcription factors and adaptor proteins. Further, M1 and M2 phenotypes play distinctive roles in cell growth and progression of inflammation-related diseases such as sepsis, obesity, cancer, and multiple sclerosis. Hence, miRNAs that modulate macrophage polarization may have therapeutic potential in the treatment of inflammation-related diseases. This review highlights recent findings in miRNA expression profiles in polarized macrophages from murine and human sources, and summarizes how these miRNAs regulate macrophage polarization. Last, therapeutic potential of miRNAs in inflammation-related diseases through modulation of macrophage polarization is also discussed.
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              Risk factors and burden of osteoarthritis.

              Osteoarthritis (OA) is one of the most common joint disorders worldwide. Its prevalence is increasing because of the growing aging of the population in developed and developing countries as well as an increase in risk factors leading to OA, particularly obesity and a sedentary lifestyle. Risk factors of OA can be divided into person-level factors (age, gender, obesity, genetics and diet) and joint-level factors (injury, malalignment and abnormal loading of the joints) that interact in a complex manner. OA is the 11th cause of disability in the world. It is responsible for activity limitations, particularly walking, and affects participation and quality of life. Patients with OA are at greater risk of all-cause mortality, particularly for cardiovascular diseases, than the general population. This excess mortality is closely associated with disability level. Consequently, strategies to reduce burden through primary and secondary prevention programs are increasingly important.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                05 March 2019
                March 2019
                : 20
                : 5
                : 1108
                Affiliations
                [1 ]IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, 20161 Milan, Italy; carlotta.perucca@ 123456grupposandonato.it (C.P.O.); deluca.paola@ 123456grupposandonato.it (P.D.L.); alessandra.colombini@ 123456grupposandonato.it (A.C.); marco.vigano@ 123456grupposandonato.it (M.V.); gaia.lugano@ 123456grupposandonato.it (G.L.); laura.degirolamo@ 123456grupposandonato.it (L.d.G.)
                [2 ]Università degli Studi di Milano, EPIGET—Epidemiology, Epigenetics and Toxicology Lab, Department of Clinical Sciences and Community Health, 20122 Milan, Italy; valentina.bollati@ 123456unimi.it
                Author notes
                [* ]Correspondence: enrico.ragni@ 123456grupposandonato.it ; Tel.: +39-02-6621-4067
                Author information
                https://orcid.org/0000-0003-0272-7417
                https://orcid.org/0000-0002-1800-3424
                https://orcid.org/0000-0002-6463-6564
                https://orcid.org/0000-0002-3068-7339
                Article
                ijms-20-01108
                10.3390/ijms20051108
                6429322
                30841483
                0a04e724-21e5-4ec0-9716-b8fcd38127e6
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 07 February 2019
                : 28 February 2019
                Categories
                Article

                Molecular biology
                adipose-derived mesenchymal stem cells,osteoarthritis,extracellular vesicles,mirna, reference genes

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