Up-Regulation of Tmevpg1 and Rmrp LncRNA Levels in Splenocytes and Brain of Mouse with Experimental Autoimmune Encephalomyelitis

Novelty in Biomedicine Original Article Up-Regulation of Tmevpg1 and Rmrp LncRNA Levels in Splenocytes and Brain of Mouse with Experimental Autoimmune Encephalomyelitis Farahani E1, Sotoodehnejadnematalahi F1, Mami S2, Fathollahi A3, Hajimolahoseini M3, Pouriran R4, Yeganeh F3* 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran Department of Immunology, School of Medicine, Ilam University of Medical Sciences, Ilam, Iran 3 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 4 School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Received: 11 May 2019; Accepted: 24 August 2019 2 Abstract Background: Two long noncoding (lnc) RNAs, which have been recognized as Tmevpg1/Ifng-AS1/NeST and Rmrp play indispensable roles in the differentiation of TH1 and TH17, respectively. The aim of the present scientific study was to analyze the expression levels of the aforementioned lncRNAs in experimental autoimmune encephalomyelitis (EAE) as an animal model for multiple sclerosis (MS). Materials and Methods: Initially, EAE was induced in C57BL/6 mice via immunization by using MOG peptide. The leukocyte infiltration rate and demyelination of neuronal axons were determined. Secondly, the expression levels of Tmevpg1, Rmrp, Tbx21, and Rorc were analyzed in the cultured splenocytes and brain lysates, by using Real-Time PCR assay; eventually, the levels of interferon-gamma and interleukin-17 evaluated by ELISA. Results: Gene expression analysis revealed that Rorc expression in the splenocytes of EAE mice in comparison to the controls was elevated; however, Tbx21 expression did not show any significant difference. Tmevpg1 and Rmrp levels increased in the splenocytes of EAE mice (4.48 times and 39.70 times, respectively, p = 0.0001). Besides, in the brain lysate, the entire genes that have been mentioned were higher than the controls (Tmevpg1: 3.35 times p = 0.02 and Rmrp 11.21 times, p = 0.0001). Conclusion: The marked up-regulation in Tmevpg1 and Rmrp transcripts suggested the essential roles of lncRNAs in the pathogenesis of EAE and multiple sclerosis indeed. Further investigations are necessary to evaluate the values of these lncRNAs as the target for the therapy or molecular marker for disease monitoring. Keywords: Experimental autoimmune encephalomyelitis (EAE), LncRNA, Tmevpg1, Rmrp, Rorc, Tbx21 *Corresponding Author: Farshid Yeganeh, Assistant Professor of immunology, Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Tel/Fax: (+) 98-2122439970; Email: ORCID ID: 0000-0001-5128-1840 Please cite this article as: Farahani E, Sotoodehnejadnematalahi F, Mami S, Fathollahi A, Hajimolahoseini M, Pouriran R, et al. UpRegulation of Tmevpg1 and Rmrp LncRNA Levels in Splenocytes and Brain of Mouse with Experimental Autoimmune Encephalomyelitis. Novel Biomed. 2019;7(4):246-53. Introduction Multiple NBM Sclerosis (MS) has been recognized as an autoimmune disease in the central nervous system (CNS), which affects more than 2 million individuals worldwide1. Different subsets of lymphocytes including T-helper 1 distinctly 246 Novelty in Biomedicine 2019, 4, 246-53 Up-Regulation of Tmevpg1 and Rmrp LncRNA Levels in Splenocytes and Brain … associated with glioma progression and invasion17, as well ischemic heart failure was noticed18. Recently, Zhang et al, reported that in PBMCs, which were obtained from the patients with MS, 2353 lncRNAs up-regulated and 389 lncRNAs down-regulated; which indicate their essential role in the development of MS development19. As of now, there has not been any scientific study reporting the transcription levels of Nest/Tmevpg1 and RMRP/Rmrp in MS and its animal model, Experimental autoimmune encephalomyelitis (EAE). Regarding the Tmevpg1 and Rmrp’s roles in T cell differentiation, we speculated that the abnormal expression of Tmevpg1 and Rmrp might involve in the pathogenesis of EAE. To evaluate the hypothesis, we explored the expressions of Tmevpg1, Rmrp, Tbx21, and Rorc in splenocytes and brains that were obtained from EAE mice. (TH1) cells and TH17 are engaged in crucial aspects of the process of demyelination that occurs in the central nervous systems during the courses of the disease2. In this process, TH1 cells, activate monocytes and macrophages by producing interferon-gamma (IFN-γ); additionally, TH17 cells play a complementary role in MS by producing the pathogenic cytokines, such as interleukin (IL)-17, and the recruitment of neutrophil cells into an inflammatory tissue2,3. The most important molecules in the differentiation of T-helper (TH) lymphocytes into TH1 and TH17 subtypes has identified as Tbx21 (the murine ortholog of human T-bet gene) and Rorc (the murine ortholog of human RORγt, gene), respectively4,5. The results of the previous studies have revealed that two long noncoding RNAs (lncRNAs) identified as Tmevpg1 and Rmrp to assist Tbx21 and Rorc respectively; in order to control the differentiating of TH1 and TH176, 7. LncRNAs are a newly discovered type of regulatory RNAs that exist throughout the genome8. Unlike micro-RNAs that prevent the expression of genes, lncRNAs can exert both inhibitory and enhancing effects on gene expression9. It has been indicated that Tmevpg1 (the ortholog of NeST gene in human) is expressed in TH1 cells and positively correlates with IFN-γ production in mouse and human6,10. As well, in TH17 cells, DEAD-box RNA helicase-5 (DDX5) is an indispensable activator for Rorc-associated transcription 11 responses . The interaction of DDX5 with Rorc requires a lncRNA called Rmrp12. In fact, identifying lncRNAs, which are associated with the pathogenesis of diseases through gene expression regulation, could assist in recognizing the pathogenesis of the diseases and finding new therapeutic targets. Previous studies demonstrated that the expression of NeST was significantly increased in Hashimoto's thyroiditis13 and Sjogren's syndrome14. In despite, the expression of NeST in peripheral blood mononuclear cells (PBMCs) of idiopathic thrombocytopenic purpura patients was lower than the controls15. Autosomal recessive mutations in Rmrp have been detected in cartilagehair hypoplasia (CHH)16 a syndrome characterized by short stature, sparse hair, and immunodeficiency. On the other hands, up-regulation of RMRP is NBM Farahani et al. Methods Experimental Animals and Induction of Experimental Autoimmune Encephalitis: Female C57BL/6 mice (6–8 weeks) were obtained from the Royan Institute (Iran). Animals were housed in standard polycarbonate cages. They were in a temperature-controlled room (23 ± 1°C). The room had a fixed 12 h light-dark cycle (08:00–20:00) and unlimited access to water and food. The Ethics and Research Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1396.454) approved all procedures. EAE induction was pe (...truncated)