Small non-coding RNA signature in multiple sclerosis patients after treatment with interferon-β

Bruna De Felice 0 1 Paolo Mondola 3 Anna Sasso 3 Giuseppe Orefice 3 Vincenzo Bresciamorra 3 Giovanni Vacca 3 Elio Biffali 2 Marco Borra 2 Raimondo Pannone 2 0 Department of Life Sciences, University of Naples II , Via Vivaldi 43, Caserta 81100 , Italy 1 DISTABIF, Department of of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Naples II , Via Vivaldi 43, 81100 Caserta , Italy 2 Zoological Station Anton Dohrn , Villa Comunale, Piazza Vittoria, Napoli 80121 , Italy 3 Department of Clinical Medicine and Surgery, University Federico II of Naples , Corso Umberto I, Naples 80131 , Italy Background: Non-coding small RNA molecules play pivotal roles in cellular and developmental processes by regulating gene expression at the post-transcriptional level. In human diseases, the roles of the non-coding small RNAs in specific degradation or translational suppression of the targeted mRNAs suggest a potential therapeutic approach of post-transcriptional gene silencing that targets the underlying disease etiology. The involvement of non-coding small RNAs in the pathogenesis of neurodegenerative diseases such as Alzheimer's , Parkinson's disease and Multiple Sclerosis has been demonstrated. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by chronic inflammation, demyelination and scarring as well as a broad spectrum of signs and symptoms. The current standard treatment for SM is interferon (IFN) that is less than ideal due to side effects. In this study we administered the standard IFN- treatment to Relapsing-Remitting MS patients, all responder to the therapy; then examined their sncRNA expression profiles in order to identify the ncRNAs that were associated with MS patients' response to IFN. Methods: 40 IFN treated Relapsing-Remitting MS patients were enrolled. We analyzed the composition of the entire small transcriptome by a small RNA cloning method, using peripheral blood from Relapsing-Remitting MS patients at baseline and 3 and 6 months after the start of IFN therapy. Real-time qPCR from the same patients group and from 20 additional patients was performed to profile miRNAs expression. Results: Beside the altered expression of several miRNAs, our analyses revealed the differential expression of small nucleolar RNAs and misc-RNAs.For the first time, we found that the expression level of miR-26a-5p changed related to INF- response. MiR-26a-5p expression was significantly higher in IFN- treated RRMS patients at 3 months treatment, keeping quite stable at 6 months treatments. Conclusions: Our results might provide insights into the mechanisms of action of IFN- treatment in MS and provide fundamentals for the development of new biomarkers and/or therapeutic tools. - Background Autoimmune diseases as Multiple Sclerosis (MS) are characterized by complex genetic traits and patho-mechanisms that translate into clinical heterogeneity [1,2]. Small noncoding RNAs (sncRNAs) are essential post-transcriptional gene regulation elements that are critical to immune system and neurodegenerative diseases [3-5] by affecting mRNA stability and the expression of multiple genes. Therefore, it is becoming increasingly evident that sncRNA species, as microRNAs, are associated with the development and progression of MS disease [6,7]. The current standard treatment for SM is interferon (IFN) that is less than ideal due to side effects. To improve the efficacy of treatments for MS, it is desirable to find biomarkers that allow early identification of treatment responders and foresee responder status. Thus, better understanding of miRNA and sncRNAs role in MS development and treatment, might contribute to the accumulation of data to understand MS pathogenesis as well as potential approaches for new therapeutic managing. To shed light into the mechanisms of action of IFN- treatment in MS, and provide fundamentals for the development of new biomarkers and/or therapeutic tools, we aimed to identify non-coding small RNAs expressed by IFN- responder treated MS patients. Therefore, we generated small RNA complementary DNA (srcDNA) libraries and sequenced 3000 srcDNA clones. We identified 6 mature miRNAs, 44 C/D box, 2 H/ACA box snorRNAs, 5 antisense fragment and 5 misc-RNAs classes from the peripheral blood mononuclear cell (PBMC) from RelapsingRemitting MS patients at the baseline and after 3 and 6 months IFN- treatment. Following, by Real-Time qPCR assay, we assessed, that, among the identified microRNAs, hsa-mir-26a-5p expression was significantly higher in IFN treated RRMS patients at 3 months treatment, keeping quite stable at 6 months treatments. We found, for the first time, that the expression of a specific miRNA, hsa-mir-26a5p, changed during INF- treatment in responder RRMS patients. Functional annotations of hsa-mir-26a-5p targets revealed that several genes were implicated in Glutamate Receptor Signaling pathway, which is notoriously altered in neurodegenerative diseases as MS. Methods Ethics statement We obtained ethics approval for our study from the ethics committee (also known as an Institutional Review Board) at our institution. All the participants had the capacity to consent and we obtained written informed consent from all participants involved in the study. Patients with relapsing-remitting MS and RNA isolation We enrolled 20 patients with a diagnosis of MS according to McDonald criteria [8], all responders to INF- Standard clinical criteria of response to interferon beta therapy were applied. Patients were considered responders if there was no increase in the EDSS score and no relapses during the follow-up period [9-11]. Additional 10 MS non-responder patients, which were characterized by failure to respond optimally from initiation of therapy as assessed by clinical measures, were recruited too. Patients (nave to the therapy) were either treated with interferon- and blood samples were obtained at a fixed time during the day just before treatment and 3 and 6 months after start of the therapy. Peripheral blood was obtained by venipuncture and immediately processed for isolation of PBMCs. PBMCs were isolated from EDTA blood via ficoll density gradient centrifugation. 1x107 cells were resuspended in QiazolH (Qiagen, Hilden, Germany) and stored at 80C. Total RNA was extracted using the TRIzol reagent (Invitrogen Carlsbad, CA). RNA samples were quality-checked by identification of 18S rRNA and 28S rRNA peaks via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). Small RNA libraries preparation and sequencing Construction of three small RNA libraries was carried out as previously described [12]. Total RNA (30 g) was pooled in 3 pools, each comprising RNA from peripheral blood leukocytes of 20 MS patients, at baseline and 3 and 6 months after the start of interferon- treatment. RNA pools were size fractionated on 15% Tris/Borate/EDTA urea polyacrylamide electrophoresis gel, and the small ncRNA (...truncated)