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miR-326 and miR-26a, two potential markers for diagnosis of relapse and remission phases in patient with relapsing–remitting multiple sclerosis

Gene, 2, 544, pages 128 - 133

Abstract

Background

Multiple sclerosis is an inflammatory autoimmune disease widely characterized by myelin destruction of CNS. Th-17 cells, have been demonstrated to play a crucial role in pathogenesis of MS. MicroRNAs are a new class of non-coding RNAs that participate in post-transcriptional regulation of gene expression. Previous studies have reported a potential role of various miRNAs in induction of Th-17 differentiation and progress of autoimmune diseases. In recent years, it has been shown that miR-326 and miR-26a involved in progress of Th-17 and MS disease.

Objective

To evaluate expression pattern of miR-326 and miR-26a in peripheral blood lymphocytes of relapsing–remitting MS patients during relapsing and remitting phases compared to healthy control subjects.

Materials and methods

Forty RR-MS patients of Isfahan population were diagnosed as relapsing (n = 20) or remitting phase (n = 20) patients according to clinical manifests and expression level of miR-26a and miR-326 was measured in these groups by quantitative real time PCR method compared to 20 healthy controls. In-silico molecular signaling pathway enrichment analysis was also performed on validated and predicted targets (targetome) of miR-26a by DAVID database to explore possible role of miR-26a in Th17 differentiation.

Results

We observed up-regulation of both miR-326 and miR-26a in relapsing phase of multiple sclerosis patients compared with remitting phase (pvalue = 0.0001) and healthy controls (pvalue = 0.0091). ROC curve analysis confirmed valuable and precise potential of miR-326 to discriminate between relapsing and remitting phases of multiple sclerosis with specificity and sensitivity of 100% at a proposed optimum cutoff point. Furthermore, in-silico molecular signaling pathway enrichment analysis detected TGF-β signaling pathway as one of the most statistically relevant pathway with miR-26a targetome.

Conclusion

Our results confirmed potential of miR-326 as a diagnostic biomarker to discriminate between relapsing and remitting phases of multiple sclerosis disease. Similar expression pattern to miR-326 and in-silico molecular enrichment analysis altogether suggest an inducing role of miR-26a in differentiation of pathogenic Th17 cells during pathogenesis of multiple sclerosis by targeting major components of the TGF-β signaling pathway (i.e. SMAD4 and SMAD1) and disarrangement of this signaling pathway.

Highlights

 

  • miR-326 is a diagnostic biomarker to discriminate phases of multiple sclerosis.
  • miR-26a has the same expression pattern as miR-326 in multiple sclerosis.
  • In-silico analysis showed inducing role of miR-26a in T-helper17 differentiation.

Abbreviations: AUC - area under the curve, CNS - central nervous system, DAVID - database for annotation, visualization and integrated discovery, hADSCs - human adipose tissue-derived stem cells, KEGG - Kyoto Encyclopedia of Genes and Genomes, miRNAs - microRNAs, MS - multiple sclerosis, PBLs - peripheral blood lymphocytes, PP - primary progressive, PR - progressive-relapsing, ROC - receiver operating characteristic, SP - secondary progressive.

Keywords: Relapsing–remitting multiple sclerosis, MicroRNA, Th-17 cells, miR-326, miR-26a.

1. Introduction

Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system (CNS) which is widely characterized by the brain and spinal cord myelin destruction (Bendszus and Storch-Hagenlocher, 2013 and Calabresi, 2004). It occurs most commonly in young people with more prevalence in women ( Bendszus and Storch-Hagenlocher, 2013 ). Once it befalls, MS disease causes attacks of neurological dysfunction that leads to disability. There are four courses for MS disease. Relapsing–remitting MS (RR-MS) is characterized by unpredictable acute attacks with worsening of symptoms in relapse period. Relapse usually appears to evolve over several days to week and then its manifestation is spontaneously recovered in remitting period. Approximately, 85% of MS patients represent RR-MS phenotype usually after 10 to 20 years. More than 50% of RR-MS patients ultimately will transmit into secondary progressive (SP-MS) which is characterized by exacerbation of symptoms without any recovery. Other two forms of disease namely, primary progressive (PP-MS) and progressive-relapsing (PR-MS) are more rare phenotypes with prevalence of 15 and 5 percentages in MS patients, respectively ( Bendszus and Storch-Hagenlocher, 2013 ).

Although etiology of MS disease has still unknown, it has been demonstrated that MS disease is an immune-mediated inflammatory response with infiltration of activated monocytes, T and B cells into CNS (Bendszus and Storch-Hagenlocher, 2013, Hauser and Oksenberg, 2006, and Lassmann et al, 2001). T helper-17 (Th-17) cells are a subset of CD4+T helper cells which differentiate from human naïve CD4+T cell in the presence of IL-6, IL-23 and TGF-β cytokines (Harrington et al, 2005, Lassmann et al, 2001, Park et al, 2005, and Veldhoen et al, 2006). The Th-17 cells are regarded as the producing IL-17, IL-21, IL-22 and GM-CSF cytokines that have major role in tissue injuries of various autoimmune diseases such as MS disease (Brucklacher-Waldert et al, 2009 and Tzartos et al, 2008), rheumatoid arthritis ( Jacobs et al., 2009 ), systemic lupus erythematous ( Yang et al., 2009 ), and psoriasis ( Fujishima et al., 2010 ). Obvious studies have demonstrated up-regulation of Th-17 cells and IL-17 level in peripheral blood mononuclear cells, cerebrospinal fluid and active lesions of multiple sclerosis patients during relapsing phases compared to remitting phase (Komiyama et al, 2006 and Tzartos et al, 2008).

MicroRNAs (miRNAs) are a new class of small non-coding RNAs which regulate gene expression post-transcriptionally by binding to 3′ UTR of their mRNA targets, and resulting in degradation or transcriptional repression of the targeted mRNA ( Bartel, 2009 ). Previous studies have reported involvement of different miRNAs in regulation of Th-17 differentiation from naïve CD4+T cells in association with pathogenesis of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Hence, these observations indicate a critical function of miRNAs in regulation of naïve T cell differentiation program into Th-17 subset (Du et al, 2009, Mycko et al, 2012, and Niimoto et al, 2010).

In one study Du et al. (2009) introduced miR-326 as a Th17-associated miRNA whose expression elevates in peripheral blood lymphocytes of multiple sclerosis patients during relapsing phase compared with remitting phase and healthy individuals, representing miR-326 correlation with the disease severity. Furthermore their in-vitro studies showed that miR-326 expression level dramatically increases during Th-17 polarizing condition by targeting ETS1, a negative regulator of Th17 differentiation ( Du et al., 2009 ). Finally Du et al. proposed valuable potential of miR-326 as a diagnostic biomarker for multiple sclerosis severity. In a different study Niimoto et al. (2010) reported up-regulation of miR-26a in peripheral blood mononuclear cells (PBMCs) and synovium tissue of 6 patients with rheumatoid arthritis in comparison with healthy subjects. In addition they revealed that the expression level of miR-26a was increased in IL-17 producing T cells during in-vitro polarization of these cells from naïve CD4+T cells, nominating miR-26a as another possible Th17-associated miRNA.

Discrimination between two periods of relapse and remitting is important especially for analysis of the progress of MS disease and effectiveness of drug therapy evaluation in patients with RR-MS in medical laboratories. Therefore, the aim of the present study was the evaluation of miR-326 and miR-26a levels in PBLs of RR-MS patients in relapse and remitting periods separately.

2. Materials and methods

2.1. Patients and samples

Forty relapsing–remitting MS (RR-MS) patients including 20 patients in relapsing phase and 20 patients in remitting phase were diagnosed based on McDonald criteria in Multiple Sclerosis Clinic of Al-Zahra Hospital (Isfahan) during a course of 8 months. After taking clearly informed consent, 5 ml of their blood was collected in EDTA containing tubes. This was accompanied by collecting 20 healthy blood samples without any infectious or allergic diseases which result in active immune system. Immediately after obtaining blood samples, they were transferred to a laboratory on ice for downstream analysis. All relapsing phase patients were new RR-MS cases with a severe attack, and without any previous consumption of immunomodulatory drugs, whereas inevitably all remitting phase patients were using immunomodulatory β-interferon. However for all remitting patients, drug adjustment was performed in such a way that blood taking was accomplished one week after previous interferon injection and just before the next shot, when the amount of drug and so its effect were in its minimal state. All patients had disease grade (SDSS) of 1. Other routine clinical information and variables of patients comprising sex, age, disease duration, MRI results and symptoms at the onset of MS are represented in Supplementary Table 1 .

2.2. PBL isolation from blood

Immediately after collecting blood samples, peripheral blood lymphocytes (PBLs) were isolated by density gradient lymphoprep (STEMCELL Technologies, USA) according to manufacturer protocol. Briefly for each sample, 3 ml of blood was diluted in proportion of 1:1 with physiological saline and was gently poured over the top of 3 ml lymphoprep solution gradient in a falcon tube. Prepared falcons were centrifuged at 800 gfor 30 min at room temperature. Finally PBLs were removed from interface phase into a 2 mlRNAase-free microtube. After washing and cell counting, PBLs were pelleted by centrifugation for 10 min at 250 gand then were frozen at − 20 °C for further RNA extraction step.

2.3. RNA extraction

Total RNA extraction from PBLs was accomplished using TRizol reagent (Invitrogen, USA) based on manufacturer instruction. Quality of extracted total RNA was determined according to 260/280 absorbance ratio, measured by Nano Drop spectrometer (Thermo Scientific, USA). In order to eliminate any potential contamination with unwanted DNA, total RNA samples undergone RNA-freeDNase(TaKaRa, Japan) treatment.

2.4. cDNA synthesis and real-time PCR

cDNA synthesis for miR-326 and miR-26a was fulfilled using a “universal cDNA synthesis kit” (Exiqon, Denmark) in poly A tailing manner, according to manufacturer leaflet. Real-time quantitative PCR reactions were carried out as triplicate by using standard protocols with an ABI PRISM 7500 instrument (Applied Biosystems, USA). Concisely, in a total volume of 10 μl, 20 ng/μl of cDNA products was added to a master mix comprising 10 pmol/μl of each miR-326 or miR-26a DNA primers (Exeqon, Denmark) and 2 U of SYBR premix ExTaqII (TaKaRa, Japan). The run method program was set as 95 °C for 5 min followed by 40 cycles of 95 °C for 5 s, 60 °C for 20 s and 72 °C for 30 s.

2.5. Statistical analysis

Real-time PCR data analysis was performed using the ∆∆CT method in Microsoft office excel 2007 software and final data were normalized by small nuclear RNA, RNU48, expression level as an endogenous control ( Fenoglio et al., 2011 ). All statistical tests were implemented by GraphPad Prism statistical software, version 5.01 (GraphPad, USA). Statistical ANOVA test was used and followed by analyzing with non-parametric Mann–Whitney post-hoct-test. To evaluate and assess the potential of miR-326 as a diagnostic indicator of disease severity to differentiate between multiple sclerosis relapsing and remitting phases, receiver operating characteristic (ROC) curves were created and the area under the curve (AUC) was measured by computing sensitivity and specificity for each possible cutoff point of the miR-326 expression level. The optimized cutoff point with consideration of 100% sensitivity and 100% specificity was chosen for drawing the ROC curve ( Supplementary Table 2 ). For all tests apvalue < 0.05 was considered statistically significant.

2.6. Molecular signaling pathway enrichment analysis

In order to perform molecular enrichment analysis on miR-26a targetome and decipher most statistically related signaling pathways that they might be involved, several in-silico online databases were used. Briefly miRwalk ( Dweep et al., 2011 ) and miRTarBase ( Hsu et al., 2011 ) databases were employed to retrieve predicted and validated targets of miR-26a. miRwalk database provides a facility for integrative prediction analysis by combining prediction results of ten prediction databases with different prediction algorithms. Then their expression in lymph nodes and leucocytes was verified by using in-silico Unigene database ( http://www.ncbi.nlm.nih.gov/unigene/ ) to decipher which of them is expressed in leukocytes or lymph nodes as it'll be substantial if they participate in T helper differentiation. Finally to carry out signaling pathway enrichment analysis, miR-26a targetome expressed in lymph node and leucocytes was imputed in the database for annotation, visualization and integrated discovery (DAVID) online database, version 6.7 ( Da Wei Huang and Lempicki, 2008 ). This database automatically outputs the results from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis ( Kanehisa and Goto, 2000 ) to identify the most statistically relevant signaling pathways and molecular networks with miR-26a targetome.

3. Results

3.1. Up-regulation of miR-326 in relapsing phase of multiple sclerosis and its potential as a relapsing phase discriminating biomarker

The expression pattern of miR-326 was evaluated by quantitative real time PCR method in three groups including RR-MS patients in relapsing phase (n = 20), RR-MS patients in remitting phase (n = 20) and a group of 20 healthy subjects. Clinical features of patients are shown in Supplementary Table 1 . Expression data of the target miRNAs were normalized with a corresponding mean value of endogenous gene small nuclear RNA, RNU48, previously confirmed as an appropriate reference gene in same conditions ( Fenoglio et al., 2011 ). miR-326 expression level was significantly elevated in the relapsing phase patient group compared with remitting phase patients (pvalue = 0.0001, approximately 4.4 fold) and healthy (pvalue = 0.0091, approximately 4.3 fold) groups. No statistically significant difference was observed between remitting phase patients and healthy subjects (pvalue = 0.3976). In addition, no significant difference was observed in miR-326 expression level of all patients together (including remitting and relapsing patients) compared to the healthy group (pvalue = 0.3421) ( Fig. 1 A). To explore the potential of miR-326 expression level as a diagnostic biomarker for discriminating relapsing and remitting phases of multiple sclerosis, we compared expression level of miR-326 in PBLs from corresponding groups by ROC analysis. ROC analysis determined the optimal cutoff value of 2.96 for miR-326 relative expression to discriminate between relapsing and remitting RR-MS patients with sensitivity and specificity of 100% and an area under the ROC curve of 1 ( Fig. 2 ).

gr1

Fig. 1 Up-regulation of miR-326 and miR-26a in relapsing phase of RR-MS patients. Quantitative PCR analysis of miR-326 expression level in PBMCs of RR-MS patients in relapsing phase (n = 20), remitting phase (n = 20) and normal controls (n = 20) (A). Quantitative PCR analysis of miR-26a in the same three groups (B). Results are normalized to those of controls and are represented relative to expression of the small nuclear RNA RNU48. *p < 0.05, **p < 0.01 and ***p < 0.005, versus control (non-parametric Mann–Whitneyt-test).

gr2

Fig. 2 ROC curve of RR-MS sample sets (relapsing and remitting patients) analyzed for relative expression level of miR-236 in peripheral blood mononuclear cells. These analyses determined the optimal cutoff value of 2.96 for miR-326 relative expression level to discriminate between relapsing and remitting RR-MS patients with sensitivity and specificity of 100% and an area under the ROC curve (AUC) of 1 (for more details please refer to the Supplementary Table 1).

3.2. Up-regulation of miR26a in relapsing phase of multiple sclerosis compared to remitting phase and healthy subjects and its similar expression pattern to miR-326

Investigating the miR-26a expression level was conducted by quantitative real time PCR, followed by statistical analysis with independent Mann–Whitneyt-test in the same three groups. It exhibited an average of 4.5 fold up-regulation in relapsing phase of multiple sclerosis patients compared with remitting phase (pvalue = 0.0001) and healthy specimens (pvalue = 0.0091). However no statistical significant differences were observed either between remitting phases and healthy subjects or all patients (including remitting and relapsing patients) and healthy group (pvalue = 0.3941) ( Fig. 1 B). Interestingly, miR-26a expression pattern was greatly resembled to what we observed for miR-326 (compare Fig. 1 A and B) indicating high tendency between miR-26a and miR-326 expression changes during relapsing or remitting phases of multiple sclerosis patients.

3.3. Molecular signaling pathway enrichment analysis of miR-26a targetome suggests possible inducing role of miR-26a in Th17 differentiation and pathogenesis through negative regulation of the TGF-β signaling pathway

To understand how possibly miR-26a is related to Th17 lineage differentiation, we performed molecular signaling pathway enrichment analysis as already described in the Materials and methods .

Using miRTarBase and miRwalk databases, 32 and 273 mRNAs were specified as validated and predicted targets of miR-26a, respectively (Supplementary Table 3, S1). All predicted targets determined by integrative prediction analysis in the miRwalk database were approved by at least 7 prediction databases. Furthermore all validated targets recovered from the miRTarBase database were supported by strong experimental evidence such as reporter assay, western blot and quantitative real time PCR. Interestingly, 19 of the 32 validated mRNAs (about 60%) were also existing in predicted mRNA list which were confirmed by at least seven prediction databases, indicating reliable threshold of 7 databases for selecting predicted mRNA targets (Supplementary Table 3, S2). However, only 18 of the validated and 138 of the predicted mRNA targets were reported to be expressed in lymph nodes by the Unigene database and these targets were selected as miR-26a targetome for further molecular enrichment analysis (Supplementary Table 3, S3).

Imputing Entrez IDs of selected miR-26a targetome into a functional annotation tool of DAVID determined statistically significant association of imputed genes with several KEGG signaling pathways ( Table 1 ). The set of imputed genes as miR-26a targetome was mostly associated and enriched in several KEGG pathways including cell cycle, Wnt signaling, MAPK signaling pathway, adherent junction, prostate cancer and interestingly TGF-β signaling pathways ranked as top related signaling pathways ( Table 1 ). The KEGG TGF-β signaling pathway is shown in Fig. 3 and targets of miR-26a are specified with red star marks. Interestingly, among these targets SMAD1 and SMAD4 are validated targets of miR-26a reported by experimental evidences.

Table 1 Top 19 most statistically relevant KEGG signaling pathways with miR-26a targetome (DAVID database).

Rank KEGG pathway The number of genes in the pathway Statistical p value
1 hsa 04110: Cell cycle 11 1.3E− 7
2 hsa 04310: Wnt signaling pathway 10 7.3E− 6
3 hsa 04520: Adherens junction 6 6.5E− 4
4 hsa 05215: Prostate cancer 6 1.3E− 3
5 hsa 05200: Pathways in cancer 10 2.7E− 3
6 hsa 04350: TGF-beta signaling pathway 5 8.1E− 3
7 hsa 04010: MAPK signaling pathway 8 1.1E− 2
8 hsa 05213: Endometrial cancer 4 1.2E− 2
9 hsa 04720: Long-term potentiation 4 2.4E− 2
10 hsa 04115: p53 signaling pathway 4 2.4E− 2
11 hsa 05211: Renal cell carcinoma 4 2.6E− 2
12 hsa 05218: Melanoma 4 2.7E− 2
13 hsa 05220: Chronic myeloid leukemia 4 3.1E− 2
14 hsa 05222: Small cell lung cancer 4 4.2E− 2
15 hsa 05210: Colorectal cancer 4 4.2E− 2
16 has 04630: Jak–STAT signaling pathway 5 5.4E− 2
17 has 04916: Melanogenesis 4 6.3E− 2
18 has 04660: T cell receptor signaling pathway 4 7.7E− 2
19 has 05221: Acute myeloid leukemia 3 1.0E− 1
gr3

Fig. 3 Involvement of miR-26a targetome in TGF-β signaling pathway. Partial diagrammatic illustration of TGF-β signaling pathway deduced from KEGG is shown. miR-26a target genes are determined by red star marks. It seems that miR-26a can inhibit TGF-β signaling pathway by targeting some positive regulators of this signaling pathway such as SMAD1, SMAD4 and P300. For more information please see the KEGG database web site: http://www.genome.jp/dbget-bin/www_bget?map04350 .

4. Discussion

Du et al. (2009) study proposed miR-326 as a valuable diagnostic biomarker of relapsing phase and severity of multiple sclerosis disease. This idea was further reviewed by Martin et al. who stated that such diagnostic biomarker would be incredibly valuable due to difficulties and challenges in collecting other target tissues (CNS or cerebral fluid) from multiple sclerosis patients, while peripheral blood is more convenient to obtain ( Martin et al., 2009 ). However, obviously more studies are needed aiming to confirm miR-326 as a biomarker of multiple sclerosis severity or relapsing state. In the current study, consistent with what was previously reported by Du et al., miR-326 expression level was significantly elevated in the relapsing phase patient group compared with remitting phase patients and healthy groups. Also, as Du et al. represented, no statistically significant difference was observed between remitting phase patients and healthy subjects. However, in contrast to Du et al. study, no significant difference was observed in miR-326 expression level of all patients (including remitting and relapsing patients) compared to the healthy group. This could be due to either limited number of patients and healthy subjects in our study or immunomodulatory drug treatment in remitting phase patients which probably could influence Th17-associated gene expression, although the adjustment of drug treatment was conducted for all remitting phase patients. ROC analysis determined the optimal cutoff value of 2.96 for miR-326 relative expression to discriminate between relapsing and remitting RR-MS patients with sensitivity and specificity of 100% and an area under the ROC curve of 1 by considering the criteria which were already described in the Materials and methods ( Fig. 2 , Supplementary Table 2 ). These results strongly suggest the discriminating potency of miR-326 expression level as a valuable and precise biomarker of relapsing phase of MS patients in our studied groups. However, it's apparent that more studies in larger scales are needed to confirm this expression cutoff point for clinical use of miR-326 as a diagnosis biomarker for relapse period from remitting period in RR-MS patients.

In a previous study, Niimoto et al. detected significantly high level of miR-26a expression in PBMCs of rheumatoid arthritis patients compared with healthy subjects. Furthermore they showed that miR-26a along with five other miRNAs (146a, 146b, 150, 155 and let-7a) is significantly upregulated in IL-17 producing cells under an in vitro Th17 polarizing condition, stating the possible promoting role of miR-26a in Th17 differentiation ( Niimoto et al., 2010 ). In the current study for the first time and to the best of our knowledge we examined miR-26a expression level as a nominated Th17-associated miRNA in correlation with miR-326 expression as a validated Th17-associated miRNA in PBMCs of remitting–relapsing multiple sclerosis patients compared with healthy subjects. Resembling an miR-326 expression pattern, we observed an average of 4.5 fold up-regulation of miR-26a in relapsing phase of multiple sclerosis, where there is high population of Th17 cells in PBMCs compared with remitting phase and healthy specimens. Taken together these results strongly confirmed possible inducing role of miR-26a in Th17 differentiation during multiple sclerosis pathogenesis in consistence with what Niimoto et al. observed in rheumatoid arthritis patients.

After discovery of Th17 subset, an inordinate number of studies were performed aiming to determine the factors and cytokines involved in Th17 lineage differentiation. In 2006 for the first time, Veldhoen et al. (2006) reported that cytokine TGF-β in context of other cytokines is essential for de-novo differentiation of Th17 subset in mice. Although now it is widely perceived that murine Th17 differentiation occurs in the presence IL-6 and TGF-β and then these cells are stabilized and amplified by IL-23 and IL-21, there is still huge controversies about the precise role of TGF-β in human Th17 differentiation ( Murdaca et al., 2011 ). While several studies have denied necessity of TGF-β for human Th17 lineage (Acosta-Rodriguez et al, 2007 and Annunziato and Romagnani, 2009), others have represented evidences that in addition to IL-1b and IL-6, the presence of TGF-β is required for human Th17 differentiation (Manel et al, 2008 and Yang et al, 2008). On the other hand it is documented that TGF-β along with IL-2 induces regulatory T cell (iTreg) lineage differentiation (Davidson et al, 2007, Huter et al, 2008, and Shevach et al, 2008), putting more confusion into the precise role of TGF-β in human CD4+T cell differentiation. However, recently Peters et al. argued that not all Th17 cells are pathogenic and reviewed key factors that discriminate between pathogenic and non-pathogenic Th17 cells in association with autoimmune disease ( Davidson et al., 2007 ). According to accomplished studies, they proposed that in the presence of TGF-β signaling, more non-pathogenic Th17 cells are induced secreting anti-inflammatory IL-10 cytokine, while the absence of TGF-β signaling results in differentiation of pathogenic Th17 cells characterized by high production of pro-inflammatory GM-CSF and even Th1-associated IFN-γ cytokines ( Davidson et al., 2007 ).

In present study we observed high expression level of miR-26a in relapse phase of multiple sclerosis patients. Niimoto et al. previously reported that miR-26a expression level significantly increases in PBMCs and synovial tissues of rheumatoid arthritis patients. They also showed that miR-26a expression level increases dramatically during in vitro differentiation of Th17 cells. It can be concluded from Fig. 3 that miR-26a can suppress TGF-β signaling pathway by targeting major contributors of this pathway comprising SMAD1, SMAD4 and P300. Interestingly, this idea has previously been proven by Luzi et al. (2008) during osteogenic differentiation of human adipose tissue-derived stem cells (hADSCs) where miR-26a may have an inducing role in osteogenic differentiation by targeting SMAD1 and affecting TGF-β signaling pathway. Hence, our molecular enrichment analysis showed statistical relevance of the miR-26a targetome with the TGF-β signaling pathway. Taken together and based on the proposed theory by Peter et al., it seems very possible that miR-26a may induce pathogenic Th17 differentiation by disarranging the TGF-β signaling pathway through targeting major components of this signaling pathway (i.e. SMAD1 and SMAD4). However more experimental loss and gain of function studies are required to confirm this theory.

5. Conclusion

In this study we have investigated enhanced transcript levels of miR-326 and miR-26a in relapsing phase of MS compared to remitting and healthy individuals. We showed a reliable cutoff point for the expression level of miR326 in order to discriminate the relapse and remitting states in MS patients. Therefore, miR-326 levels could be used as a diagnostic indicator of relapsing phase of RR-MS patients. However more studies in larger scales are needed to confirm this notion. Furthermore, we observed an increased rate of miR26a transcripts in MS patients which showed the similarity with enhanced expression of miR326. We also examined the potential role of miR-26a in Th17 differentiation by performing in-silico molecular enrichment analysis. These analyses strongly suggested the inducing role of miR-26a in differentiation of pathogenic Th17 by targeting major factors of TGF-β signaling and so disarranging this signaling pathway. However this hypothesis awaits in vivo and in vitro experimental validation.

Taken together, we propose that miR-26a and miR-326, as two diagnostic biomarkers could be used not only for detection of relapse period but also for effectiveness of drug therapy evaluation in patients with RR-MS in medical libraries. Meanwhile, these two micro-RNAs could be pinpointed as two therapeutic targets for prevention of MS disease progress.

The following are the supplementary data related to this article.

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Supplementary Table 1 Patient's clinical characteristics and variables.

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Supplementary Table 2 Detailed report of sensitivity and specificity.

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Supplementary tables.

Author contributions

Mohammad Amin Honardoost: Experimental design, collection and/or assembly of data, data analysis, interpretation, and manuscript writing.

Abbas Kiani-Esfahani: Experimental design, data analysis and interpretation.

Kamran Ghaedi: Conception and design, financial support, data analysis, interpretation, manuscript writing, and final approval of the manuscript.

Masoud Etemadifar: Conception, design, data analysis, interpretation, financial support, and final approval of the manuscript.

Mansoor Salehi: Conception, design, data analysis, interpretation, financial support and final approval of the manuscript.

Conflict of interest

None of the authors has any conflicts of interest to disclose and all authors support submission to this journal.

Acknowledgments

This study was funded partly by a grant-in-aid of research from Isfahan University of Medical Sciences to Mansoor Salehi (Ph.D.) and partly by University of Isfahan in support of Mohammad Amin Honardoost for obtaining his M.Sc. degree from the University of Isfahan.

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Footnotes

a Division of Cellular and Molecular Biology, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran

b Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

c Cellular & Molecular Immunology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

d Department of Neurology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

e Multiple Sclerosis and Neuroimmunology Research Center, Isfahan, Iran

f Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

lowast Correspondence to: K. Ghaedi, Division of Cellular and Molecular Biology, Department of Biology, Faculty of Sciences, University of Isfahan, 81746-73441 Isfahan, Iran.

lowastlowast Correspondence to: M. Salehi, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, 81746-73461 Isfahan, Iran.