Original Article
 

Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4

Abstract

Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes.
EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively.
MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells.
Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

1. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215-33.
2. Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008;9(2):102-14.
3. Paschon V, Takada SH, Ikebara JM, Sousa E, Raeisossadati R, Ulrich H, et al. Interplay Between Exosomes, microRNAs and Toll-Like Receptors in Brain Disorders. Mol Neurobiol. 2016;53(3):2016-28.
4. Constantinescu CS, Farooqi N, O'Brien K, Gran B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). Br J Pharmacol. 2011;164(4):1079-106.
5. Farooqi N, Gran B, Constantinescu CS. Are current disease-modifying therapeutics in multiple sclerosis justified on the basis of studies in experimental autoimmune encephalomyelitis? J Neurochem. 2010;115(4):829-44.
6. Jiang Z, Jiang JX, Zhang GX. Macrophages: a double-edged sword in experimental autoimmune encephalomyelitis. Immunol Lett. 2014;160(1):17-22.
7. Tufekci KU, Oner MG, Genc S, Genc K. MicroRNAs and Multiple Sclerosis. Autoimmune Diseases. 2011;2011:807426.
8. Jagot F, Davoust N. Is It worth Considering Circulating microRNAs in Multiple Sclerosis? Front Immunol. 2016;7:129.
9. Junker A, Hohlfeld R, Meinl E. The emerging role of microRNAs in multiple sclerosis. Nat Rev Neurol. 2011;7(1):56-9.
10. Gandhi R. miRNA in multiple sclerosis: search for novel biomarkers. Mult Scler. 2015;21(9):1095-103.
11. Junker A, Krumbholz M, Eisele S, Mohan H, Augstein F, Bittner R, et al. MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47. Brain. 2009;132(Pt 12):3342-52.
12. Sarkozy M, Kahan Z, Csont T. A myriad of roles of miR-25 in health and disease. Oncotarget. 2018;9(30):21580-612.
13. Noorbakhsh F, Ellestad KK, Maingat F, Warren KG, Han MH, Steinman L, et al. Impaired neurosteroid synthesis in multiple sclerosis. Brain. 2011;134(Pt 9):2703-21.
14. Nuzziello N, Vilardo L, Pelucchi P, Consiglio A, Liuni S, Trojano M, et al. Investigating the Role of MicroRNA and Transcription Factor Co-regulatory Networks in Multiple Sclerosis Pathogenesis. Int J Mol Sci. 2018;19(11).
15. De Santis G, Ferracin M, Biondani A, Caniatti L, Rosaria Tola M, Castellazzi M, et al. Altered miRNA expression in T regulatory cells in course of multiple sclerosis. J Neuroimmunol. 2010;226(1-2):165-71.
16. Petrocca F, Vecchione A, Croce CM. Emerging role of miR-106b-25/miR-17-92 clusters in the control of transforming growth factor beta signaling. Cancer Res. 2008;68(20):8191-4.
17. Giuliani F, Metz LM, Wilson T, Fan Y, Bar-Or A, Yong VW. Additive effect of the combination of glatiramer acetate and minocycline in a model of MS. J Neuroimmunol. 2005;158(1-2):213-21.
18. Tsutsui S, Schnermann J, Noorbakhsh F, Henry S, Yong VW, Winston BW, et al. A1 adenosine receptor upregulation and activation attenuates neuroinflammation and demyelination in a model of multiple sclerosis. J Neurosci. 2004;24(6):1521-9.
19. Eng LF, Ghirnikar RS, Lee YL. Inflammation in EAE: role of chemokine/cytokine expression by resident and infiltrating cells. Neurochem Res. 1996;21(4):511-25.
20. Schellenberg AE, Buist R, Yong VW, Del Bigio MR, Peeling J. Magnetic resonance imaging of blood-spinal cord barrier disruption in mice with experimental autoimmune encephalomyelitis. Magn Reson Med. 2007;58(2):298-305.
21. Sievers C, Meira M, Hoffmann F, Fontoura P, Kappos L, Lindberg RL. Altered microRNA expression in B lymphocytes in multiple sclerosis: towards a better understanding of treatment effects. Clin Immunol. 2012;144(1):70-9.
22. Liguori M, Nuzziello N, Licciulli F, Consiglio A, Simone M, Viterbo RG, et al. Combined microRNA and mRNA expression analysis in pediatric multiple sclerosis: an integrated approach to uncover novel pathogenic mechanisms of the disease. Hum Mol Genet. 2018;27(1):66-79.
23. Finardi A, Diceglie M, Carbone L, Arno C, Mandelli A, De Santis G, et al. Mir106b-25 and Mir17-92 Are Crucially Involved in the Development of Experimental Neuroinflammation. Front Neurol. 2020;11:912.
24. Ramesh S, Qi XJ, Wildey GM, Robinson J, Molkentin J, Letterio J, et al. TGF beta-mediated BIM expression and apoptosis are regulated through SMAD3-dependent expression of the MAPK phosphatase MKP2. EMBO Rep. 2008;9(10):990-7.
25. Zhang H, Zuo Z, Lu X, Wang L, Wang H, Zhu Z. MiR-25 regulates apoptosis by targeting Bim in human ovarian cancer. Oncol Rep. 2012;27(2):594-8.
26. Worby CA, Dixon JE. Pten. Annu Rev Biochem. 2014;83:641-69.
27. Li B, Wang H, Guan J, Xiong X, Chen L. Experimental study of miR-25 targeting PTEN/PI3K/AKT signalling pathway to inhibit apoptosis of ovarian cancer SKOV3 cells. Biomedical Res. 2017; 28(14).
28. Kwak YG, Song CH, Yi HK, Hwang PH, Kim JS, Lee KS, et al. Involvement of PTEN in airway hyperresponsiveness and inflammation in bronchial asthma. J Clin Invest. 2003;111(7):1083-92.
29. Lee KS, Park SJ, Hwang PH, Yi HK, Song CH, Chai OH, et al. PPAR-gamma modulates allergic inflammation through up-regulation of PTEN. FASEB J. 2005;19(8):1033-5.
30. Kim HS, Jang SW, Lee W, Kim K, Sohn H, Hwang SS, et al. PTEN drives Th17 cell differentiation by preventing IL-2 production. J Exp Med. 2017;214(11):3381-98.
31. Ghaleb AM, Yang VW. Kruppel-like factor 4 (KLF4): What we currently know. Gene. 2017;611:27-37.
32. Zheng B, Han M, Bernier M, Zhang XH, Meng F, Miao SB, et al. Kruppel-like factor 4 inhibits proliferation by platelet-derived growth factor receptor beta-mediated, not by retinoic acid receptor alpha-mediated, phosphatidylinositol 3-kinase and ERK signaling in vascular smooth muscle cells. J Biol Chem. 2009;284(34):22773-85.
33. Hartmann P, Zhou Z, Natarelli L, Wei Y, Nazari-Jahantigh M, Zhu M, et al. Endothelial Dicer promotes atherosclerosis and vascular inflammation by miRNA-103-mediated suppression of KLF4. Nat Commun. 2016;7(3):10521-8.
34. Li Z, Jia Y, Han S, Wang X, Han F, Zhang J, et al. Klf4 Alleviates Lipopolysaccharide-Induced Inflammation by Inducing Expression of MCP-1 Induced Protein 1 to Deubiquitinate TRAF6. Cell Physiol Biochem. 2018;47(6):2278-90.
35. Zhang X, Wang L, Han Z, Dong J, Pang D, Fu Y, et al. KLF4 alleviates cerebral vascular injury by ameliorating vascular endothelial inflammation and regulating tight junction protein expression following ischemic stroke. J Neuroinflammation. 2020;17(1):107.
36. Tetreault MP, Wang ML, Yang Y, Travis J, Yu QC, Klein-Szanto AJ, et al. Klf4 overexpression activates epithelial cytokines and inflammation-mediated esophageal squamous cell cancer in mice. Gastroenterology. 2010;139(6):2124-34 e9.
37. Shaverdashvili K, Padlo J, Weinblatt D, Jia Y, Jiang W, Rao D, et al. KLF4 activates NFkappaB signaling and esophageal epithelial inflammation via the Rho-related GTP-binding protein RHOF. PLoS One. 2019;14(4):e0215746.
38. Ahn M, Kim J, Park C, Jung K, Moon C, Shin T. Immunohistochemical study of Kruppel-like factor 4 in the spinal cords of rats with experimental autoimmune encephalomyelitis. Acta Histochem. 2015;117(6):521-7.
39. An J, Golech S, Klaewsongkram J, Zhang Y, Subedi K, Huston GE, et al. Kruppel-like factor 4 (KLF4) directly regulates proliferation in thymocyte development and IL-17 expression during Th17 differentiation. FASEB J. 2011;25(10):3634-45.
40. Hao Z, Sheng Y, Duncan GS, Li WY, Dominguez C, Sylvester J, et al. K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression. Nat Commun. 2017;8:14003.
41. Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol. 2005;23:683-747.
42. Dendrou CA, Fugger L, Friese MA. Immunopathology of multiple sclerosis. Nat Rev Immunol. 2015;15(9):545-58.
43. Noorbakhsh F, Tsutsui S, Vergnolle N, Boven LA, Shariat N, Vodjgani M, et al. Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis. J Exp Med. 2006;203(2):425-35.
44. Casadei L, Calore F, Creighton CJ, Guescini M, Batte K, Iwenofu OH, et al. Exosome-Derived miR-25-3p and miR-92a-3p Stimulate Liposarcoma Progression. Cancer Res. 2017;77(14):3846-56.
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IssueVol 20 No 3 (2021) QRcode
SectionOriginal Article(s)
Published2021-06-06
DOI https://doi.org/10.18502/ijaai.v20i3.6337
Keywords
Autoimmune diseases of the nervous system Encephalomyelitis MicroRNAs Multiple sclerosis

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How to Cite
1.
Zare-Chahoki A, Ahmadi-Zeidabadi M, Azadarmaki S, Ghorbani S, Noorbakhsh F. Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4. Iran J Allergy Asthma Immunol. 20(3):314-325.