Abstract

Background

   One of the main issues in pathogenesis of MS is Th17/Treg imbalance.
   There are growing interests in nominating miRNAs involved in Th17 cell
   differentiation, suggesting them as new therapeutic agents that may
   reduce progression of different autoimmune diseases specially MS.

Objectives

   We assessed transcript levels of miR-141 and miR-200a in MS patients,
   during relapsing and remitting phases. We also investigated possible
   role of miR-141, miR-200a in inducing differentiation to Th17 cells.

Materials and Methods

   Forty RR-MS patient samples including relapsing (n=20) and remitting
   (n=20) phases were chosen. Expression level of miR-141 and miR-200a
   were measured by RT-q PCR and compared to healthy control group (n=10).
   In-silico analyses on miR-141 and miR-200a targetome showed involvement
   of both miRNAs in T helper cell differentiation pathways including
   TGF-β, mTOR and JAK/STAT.

Results

   We observed that percentage of RORγt^+ CD4^+ T cells increase in
   relapsing phase while FOXP3^+ CD4^+ increase in remitting phase of MS
   patients. Furthermore, both miR-141 and miR-200a show up-regulation in
   relapsing phase of MS patients compared to remitting and control
   groups. Interestingly, expression level of target genes of miR-141 and
   miR-200a, which were assessed through in-silico methods, show
   down-regulation in relapsing phase of MS patients.

Conclusions

   According to our results, miR-141 and miR-200a may be key miRNAs in
   progression of symptoms of MS through inducing differentiation of Th17
   cells and inhibiting differentiation to Treg cells. Our data suggest
   that these miRNAs may probably inhibit negative regulators of Th17 cell
   differentiation, thus promoting its differentiation.

Introduction

   Multiple Sclerosis (MS) is a neurodegenerative chronic autoimmune
   disease of the CNS in which myelin and axons are destroyed to different
   degrees [[33]1]. Although development of MS is highly irregular, it is
   mostly considered by occurrence of reversible neurological malfunctions
   which deteriorates over time.

   Epidemiology of MS in developing countries as Iran and large cities as
   Isfahan displays that there is a wild growth in frequency of affected
   patients with an overall prevalence of 85.8 per 100000 [[34]2]. MS is
   known to be a multifactorial disease with still no definite cause but
   it appears that combination of environmental factors, epigenetic and
   genetics lead to continuing immune attacks on the CNS [[35]3].

   Primarily it was supposed that a subset of CD4^+ T cells with a Th1
   phenotype producing IFN-γ is critical in autoimmunity of MS, however it
   is now clear that IL-17 producing CD4^+ T cells, known as Th17, are the
   main responsible cells for inflammation and pathogenesis of MS [[36]4].
   Th17 cells mainly do their effects through secreting IL-17, IL-21,
   IL-22 and GM-CSF, which are essential for autoimmune neuro-inflammation
   [[37]5,[38]6]. Generally, activation of different STAT transcription
   factors along with master regulator of each lineage leads to
   differentiation of various CD4^+ T cell subtypes. Following the
   activation of specific transcription factors of STAT3/RORγt, naïve
   CD4^+ T cells differentiate to Th17 [[39]7,[40]8]. Various studies
   indicate up-regulation of Th17 cells in different autoimmune diseases
   such as MS and experimental autoimmune encephalomyelitis (EAE).
   Furthermore, Tzartos et al. showed that expression level of IL-17
   increases in active lesions of MS patients [[41]9,[42]10]. In addition,
   as well as other autoimmune diseases, MS is closely related to
   deficiencies in immune regulation through malfunction or decreased
   number of regulatory T cells (Treg) [[43]4] and its differentiation
   occurs through activation of STAT5/ FOXP3 [[44]8].

   Various studies showed that microRNAs (miRNAs) play significant roles
   in different processes including hematopoiesis and function of diverse
   sets of immune cells such as T cells through suppressing different
   mRNAs in post-transcriptional level [[45]11,[46]12].

   miR-200 family includes two cluster of miRNAs which one is on
   chromosome 1p36.3 (miR-200a/200b/429) and its members have the same
   seed length (AAUACU (while the other one is on chromosome 12p13
   (miR-141/200c) and its members have the same seed length (AACACU) which
   is highly similar to cluster one [[47]13].

   Different studies including our previous study [[48]14], have explored
   deregulation of different miRNAs in peripheral blood mononuclear cells
   (PBMC), B cells, CD4^+ T cells and tissues of MS patients, so far.
   Furthermore, several studies have also investigated the role of
   different miRNAs on differentiation of Th17 cells. Studies on miR-141
   and miR-200a have shown that these miRNAs are involved in different
   autoimmune diseases, although their role in different cancers is well
   known as well. Studies on systemic lupus erythematosus (SLE),
   inflammatory bowel disease (IBD), psoriasis and other immune-related
   diseases display deregulation of miR-141 and miR-200a [[49]15–[50]17].
   Despite aforementioned studies, miR-141 and miR-200 were never studied
   or focused on in MS patients and T helper cell differentiation before.
   The initial notion is that in case of miR-141 and miR-200a’s role in
   differentiation of Th17 cells, they should display up-regulation
   consistent with increase in the number of Th17 cells in relapsing phase
   of MS. Inversely they should indicate down-regulation with reduction of
   Th17 cells in remitting phase.

   The aim of the current study is to evaluate miR-141 and miR-200a
   expression in CD4^+ T cells of relapsing versus remitting phase of MS
   patients; through which we tried to find their possible role in
   differentiation or suppression of Th17 and/or Treg cells by assessing
   the correlation between them and the percentage of these cells in
   different phases of MS. Furthermore, possible targets of miR-141 and
   miR-200a that could be involved in differentiation of T helper cells
   were established in-silico, and their accuracy was investigated.
   Therefore, concluding intention of this study is to suggest miR-141 and
   miR-200a as novel miRNAs involved in differentiation of Th17 cells,
   thereby pathogenesis of MS.

Materials and Methods

Patients and control samples

   Total blood samples (10 ml with EDTA) were drawn from 40 patients with
   relapsing-remitting MS (RRMS) attending Al-Zahra Hospital (Isfahan,
   Iran), including 20 newly diagnosed (as relapsing phase) and 20
   patients in remitting phase. Ten samples were also taken from healthy
   volunteers. Patients were diagnosed based on McDonald criteria. All
   patients in relapsing phase had no drug treatment while all patients in
   remitting phase were chosen among those persons who were only treated
   with Synovex (IFN-β) to avoid drug complication effects. Sampling were
   done a week after previous injection and just before the next shot to
   minimize the drug effects. Written informed consent was obtained from
   all studied subjects. All study protocols and written consent forms
   taken from the individuals in this study were approved by institutional
   review board of Royan Institute (Project Id. No. 91000573). Written
   consent forms are recorded and available in Royan institute archives.
   All clinical information of patients including age, sex, disease
   duration and MRI results are signified in [51]S1 Table.

Cell isolation

   PBMC were isolated using density gradient lymphoprep (STEMCELL
   Technologies, USA) according to manufacturer’s instruction. Cell
   subpopulations, CD4^+ T cells, were isolated from PBMC with CD4^+ T
   cell isolation kit II human of Miltenyi Biotec (Miltenyi Biotec,
   Bergisch Gladbach, Germany), with purity of more than 95 percent
   [[52]18] and according to manufacturer’s instruction.

Flow Cytometry

   To quantify RORγt and FOXP3 levels as Th17 and Treg cells markers
   respectively, flow cytometry was performed. At the first step, the
   isolated cells were fixed and stained with the following antibodies:
   Anti-Human Foxp3 PE, Anti-Human/Mouse ROR γ(t) PE, Anti-Human CD4 Alexa
   Fluor 488 and analyzed on Becton Dickinson FACSCalibur flow cytometer
   (USA). Data were analyzed by comparison of the Mouse IgG1 K Isotype
   Control PE, Rat IgG2a K Isotype Control PE, Mouse IgG2b K Isotype
   Control Alexa Fluor 488(all antibodies were purchased from eBioscience,
   USA). A forward and side scatter gate was used to select lymphocyte
   population and fluorescence compensation was set according to labeled
   lymphocytes with only green and only red fluorescence separately versus
   isotype control. Then, the samples were run in acquisition phase. Green
   fluorescence from FITC-conjugated antibodies and red fluorescence from
   PE-conjugated antibodies were collected in fluorescence detector 1 and
   2 respectively. Percentages of Th-17 and Treg cells were determined
   after gating without debris and aggregate. CellQuest Pro software was
   used to analyze data.

RNA extraction

   Total RNA (including miRNA) were isolated by TRizol reagent
   (Invitrogen,USA) according to manufacturer’s instruction.

cDNA synthesis and real-time PCR

   cDNA synthesis for miR-141 and miR-200a was achieved by using a
   “universal cDNA synthesis kit” (Exiqon, Denmark) in a poly A tailing
   method, as stated by manufacturer. Real-time quantitative PCR (RT-qPCR)
   reactions were performed using standard protocol with an ABI PRISM 7500
   instrument (Applied Biosystems, USA); cDNA products were added to a
   master mix comprising 10 pmol/μl of each miR-141 or miR-200a DNA
   primers (Exeqon, Denmark) and 2 U of SYBR premix ExTaq II (TaKaRa,
   Japan), similar to our previous study [[53]14]. RNU48 small nucleolar
   RNA was quantified as the reference to normalize differences in total
   RNA levels. cDNA synthesis for IL-17A, IL-23R, RORC, TGF-β, FOXP3,
   SMAD2, GATA3 and FOXO3 was carried out on a total RNA which has
   reverse-transcribed by RevertAid First Strand cDNA synthesis Kit
   (Thermo Scientific, USA) using random hexamer primers. RT-qPCR was
   carried out using specific primer pairs in ABI PRISM 7500 instrument
   (Applied Biosystems, USA). The expression level of these genes was
   normalized by 18srRNA as reference gene. All reactions were carried out
   in triplicate. Real-time data were evaluated and reported based on ΔΔCT
   method.

Poly Acrylamide gel & T/A cloning

   To evaluate the specificity of miR-141 and miR-200a primers, real time
   PCR products were run on 12% poly acrylamide gel to see solo amplified
   band with length of 80bp. This band was extracted from the gel and T/A
   cloned into pTZ57R/T vector (Thermo Scientific) and were send to
   sequence.

Statistical analysis

   All statistical tests were executed by SPSS (version 20). Comparison
   between groups was analyzed by statistical ANOVA test and followed by
   analyzing with nonparametric Mann—Whitney post-hoc t-test. Data are
   presented as mean ± SEM and considered significant at p < 0.05.

Molecular signaling pathway enrichment analysis

   In order to accomplish molecular enrichment analysis on miR-141 and
   miR-200a targetome and to find most related signaling pathways which
   might be involved, we used online in-silico databases of miRWalk
   [[54]18] and miRTarBase [[55]19] to attain predicted and validated
   targets of miR-141 and miR-200a respectively. We also used DIANA
   miRPath v.2.0 [[56]20] to visualize pathways of validated targets of
   miR-141 and miR-200a as a heatmap. Next, using UniGene database
   ([57]http://www.ncbi.nlm.nih.gov/unigene/) and subsequent EST profile,
   we investigated their expression in lymph node and thymus since it will
   be indispensable if they are involved in differentiation of T helper
   cells. Finally, miR-141 and miR-200a targetome expressed in lymph nodes
   and thymus were assigned to database for annotation, visualization and
   integrated discovery (DAVID) bioinformatics database [[58]21] to
   categorize the most applicable pathways and molecular networks with
   miR-141 and miR-200a targetome.

Results

Number of Th17 cells increased significantly in relapsing phase of MS, while
number of Treg cells increased in remission phase of MS

   First, to evaluate the number of Th17 and Treg cells in different
   phases of MS, part of CD4^+ T cells yielded from MACS were stained with
   FoxP3, RORγt and CD4 antibodies as explained in materials and methods.
   Results showed that percentage of RORγt^+ CD4^+ T cells significantly
   increased in relapsing versus remitting and control group (p value =
   0.0002, p value = 0.0003 respectively), while percentage of FoxP3^+
   CD4^+ T cells significantly increased in remitting compared to
   relapsing and healthy controls (p value = 0.003, p value = 0.001
   respectively) (Fig [59]1A–[60]1C).

Fig 1. Flow cytometry of FoxP3^+ CD4^+ T cells and RORɣt+ CD4+ T cells.

   [61]Fig 1
   [62]Open in a new tab

   CD4^+ T cells were isolated by CD4+ Tcell isolation kit II human of
   Miltenyi Biotec and stained with respective antibodies and evaluated in
   relapsing phase (n = 20) and remitting phase (n = 20) of MS patients
   and healthy controls (n = 10). A forward and side scatter gate was used
   to select lymphocyte population and fluorescence compensation was set
   according to labeled lymphocytes with only green and only red
   fluorescent separately versus isotype control (A). Percentage of RORγt+
   CD4+ T cells measured by Flow cytometry, shows meaningful increase in
   relapsing group (B) while percentage of FoxP3^+ CD4^+ T cells elevates
   in remitting group (C) (*p < 0.05, **p < 0.01 and ***p < 0.005,
   non-parametric Mann-Whitney t-test) (RP: Relapsing patient, MP:
   Remitting patient, HV: Healthy volunteer).

Up-regulation of miR-141 in relapsing phase of MS

   Investigation of miR-141 expression level was carried out by RT-qPCR in
   three groups including relapsing and remitting phase of MS patients and
   healthy controls. Expression of miRNAs were normalized by small
   nucleolar RNA, RNU48, which was established to be the suitable
   reference gene beforehand [[63]22]. miR-141 expression level was
   significantly higher in relapsing group compared to control and
   remitting groups (p value = 0.006 and p value = 0.001 respectively).
   Nevertheless, no meaningful difference was observed between control and
   remitting groups (p value = 0.837) ([64]Fig 2A).

Fig 2. Up-regulation of miR-141 and miR-200a in relapsing phase of MS
patients.

   Fig 2
   [65]Open in a new tab

   RT-qPCR analysis of miR-141 expression level in CD4^+ T cells of MS
   patients in relapsing phase (n = 20), remitting phase (n = 20) and
   healthy controls (n = 10) (A). RT-qPCR analysis of miR-200a expression
   level in CD4^+ T cells of MS patients in the same groups (B). Results
   are normalized relative to expression level of reference gene, RNU48
   (*p < 0.05, **p < 0.01 and ***p < 0.005, non-parametric Mann-Whitney
   t-test) (RP: Relapsing patient, MP: Remitting patient, HV: Healthy
   volunteer).

Up-regulation of miR-200a in relapsing phase of MS and its similar expression
pattern to miR-141

   Analysis of miR-200a showed that miR-200a displays the same exact
   pattern as miR-141 expression. miR-200a expression level was
   significantly elevated in relapsing group versus control and remitting
   group (p value = 0.006 and p value = 0.003 respectively). There was
   also no significant difference between control and remitting group (p
   value = 0.741) ([66]Fig 2B).

   Primer specificity for miR-141, miR-200a and RNU48 was evaluated by
   poly acrylamide gel electrophoresis ([67]S1 Fig). Additionally,
   sequencing results of cloned PCR products into pTZ57R/T
   ([68]S2A–[69]S2C Fig) confirmed the specificity of our primers.

Expression analysis of master transcription factors involved in Th17/Treg
differentiation along with IL-17A gene expression in CD4^+ T cells

   To confirm the results of flow cytometry, we analyzed whether the
   expression level of master markers of Th17&Treg cells alter during the
   change of disease course from relapsing to remitting. The expression
   level of IL-23R, RORC, TGF-β and FOXP3 were normalized to 18srRNA as
   reference gene. Combined with this, we investigated the transcript
   level of IL-17A in relapsing group versus remitting and control groups.
   Expression level of IL-17A significantly increased in relapsing phase
   compared to remitting phase (p value = 0.023) but no significant
   differences were found between remitting with control and relapsing
   with control group (p value = 0.694 and p value = 0.329 respectively).
   IL23R expression displayed significant increase in relapsing versus
   remitting (p value = 0.042), however no significance was observed in
   remitting versus control and control versus relapsing (p value = 0.815
   and p value = 0.058 respectively). Analyses of RORC expression
   demonstrated that there is a significant rise in relapsing group
   compared to both remitting and control group (p value = 0.0007 and p
   value = 0.002 respectively), but there was no significant difference
   between remitting and control groups (p value = 0.914). TGF-β
   expression level showed significant increase in relapsing versus
   control (p value = 0.008). TGF-β also up-regulated in remitting group
   versus control (p value = 0.029), however no meaningful variation was
   found between relapsing and remitting (p value = 0.573). Finally, the
   expression level of FOXP3 significantly decreased in relapsing compared
   to control group (p value = 0.046) but there were no significant
   difference between relapsing with remitting and control with remitting
   (p value = 0.341 and p value = 0.187 respectively) ([70]Fig 3).

Fig 3. The expression level of master markers of Th17& Treg cells and IL-17A
in CD4^+ T cells of relapsing group (n = 20), remitting group (n = 20) and
healthy controls (n = 10) in MS patients.

   Fig 3
   [71]Open in a new tab

   Results are normalized relative to expression level of reference gene,
   18srRNA (*p < 0.05, **p < 0.01 and ***p < 0.005, non-parametric
   Mann-Whitney t-test) (RP: Relapsing patient, MP: Remitting patient, HV:
   Healthy volunteer).

Molecular signaling pathway enrichment analysis of miR-141 and miR-200a
targetome proposes possible inducing role of miR-141 and miR-200a in
differentiation of Th17 cells

   To recognize the possible role of miR-141 and miR-200a in Th17
   differentiation, molecular signaling pathway enrichment analysis was
   conducted. Using miRWalk and miRTarBase databases, 2248 and 25
   predicted and validated mRNAs for miR-141 and 2195 and 26 predicted and
   validated mRNAs for miR-200a were specified, respectively ([72]S2A and
   [73]S2B Table). All predicted targets in miRWalk were confirmed in at
   least five prediction databases. Additionally, all validated mRNA
   targets collected from miRTarBase are supported by experimental
   evidences including RT-qPCR, western blotting and reporter assay.
   Heatmap view of validated targets of miR-141 and miR-200a designed by
   DIANA miRPath v.2.0 showed that the only pathway involved in
   differentiation of Th17 cells which miR-141 and miR-200a might
   effectively influence is mTOR signaling pathway [[74]23] ([75]Fig 4).
   Additional examination displayed that 14 of validated targets of
   miR-141 and 17 of validated targets of miR-200a were expressed in lymph
   node and thymus by Unigene database ([76]S2D and [77]S2E Table).

Fig 4. Heatmap view of pathways in which validated targets of miR-141 and
miR-200a are involved.

   [78]Fig 4
   [79]Open in a new tab

   The heatmap was drawn in consequent of enrichment analysis on miR-141
   and miR-200a targetome (for valid targets) in which a merged p-value is
   calculated for each pathway by applying Fisher’s meta-analysis manner.
   The resulting p-value represents the examined pathways that are
   significantly enriched with gene targets of miR-141 and miR-200a. Color
   gradient displays the importance of mentioned pathways. Heatmap was
   drawn based on validated targets of miR-141 and miR-200a, according to
   DIANA miRPath. Notably, mTOR signaling pathway was specified as one of
   the major pathways based on its involvement in differentiation of Th17
   cells. Analysis shows that miR-141 affects mTOR pathway more effective
   than miR-200a does.

   Moreover, 14 mRNAs of all predicted targets for miR-141 and miR-200a
   that their roles in differentiation of T helper cells have been
   revealed in our studies were specified and taken into Unigene. The
   incoming results exhibited their expression in lymph node and thymus
   ([80]S2D and [81]S2E Table). Lastly, validated and predicted genes with
   defined expression in lymph node and thymus were chosen as miR-141 and
   miR-200a targetome for extra molecular enrichment analysis. Inputting
   official gene symbols of miR-141 and miR-200a targetome into the
   functional annotation tool of DAVID, a combined view of statistically
   meaningful pathways associated with above genes in BIOCARTA, KEGG and
   PANTHER was acknowledged ([82]Table 1).

Table 1. Top statistically related signaling pathways to miR-141 and miR-200a
targetome obtained from different databases by means of DAVID tool.

   miR-141 miR-200a
   Category Pathway P value Category Pathway P value
   KEGG pathway Pathways in cancer 1.3E-4 KEGG pathway Pathways in cancer
   3.2E-4
   PANTHER pathway JAK / STAT signaling pathway 3.4E-6 PANTHER pathway JAK
   / STAT signaling pathway 4.5E-6
   PANTHER pathway TGF-β signaling pathway 6.8E-3 PANTHER pathway TGF-β
   signaling pathway 8.7E-3
   KEGG pathway Chronic myeloid leukemia 1.0E-3 PANTHER pathway
   Interleukin signaling pathway 2.6E-2
   PANTHER pathway EGF receptor signaling pathway 3.5E-2 PANTHER pathway
   EGF receptor signaling pathway 4.1E-2
   BIOCARTA mTOR Signaling Pathway 1.9E-3 PANTHER pathway PDGF signaling
   pathway 7.6E-2
   KEGG pathway Acute myeloid leukemia 1.1E-2 KEGG pathway Endometrial
   cancer 1.1E-2
   KEGG pathway Insulin signaling pathway 5.2E-2 KEGG pathway Chronic
   myeloid leukemia 2.3E-2
   PANTHER pathway Insulin/IGF pathway-protein kinase B signaling cascade
   7.6E-2 KEGG pathway Adherence junction 2.4E-2
   BIOCARTA Regulation of eIF4e and p70 S6 Kinase 2.2E-2 BIOCARTA mTOR
   Signaling Pathway 3.1E-2
   PANTHER pathway Interleukin signaling pathway 9.5E-2 KEGG pathway
   Colorectal cancer 2.8E-2
   BIOCARTA GATA3 participate in activating the Th2 cytokine genes
   expression 9.4E-2 PANTHER pathway p53 pathway feedback loops 2 4.6E-2
   KEGG pathway Insulin signaling pathway 5.2E-2 KEGG pathway Leukocyte
   transendothelial migration 5.2E-2
   PANTHER pathway Ras Pathway 8.8E-2
   BIOCARTA GATA3 participate in activating the Th2 cytokine genes 1.0E-1
   [83]Open in a new tab

   miR-141 and miR-200a targetome were commonly enriched in pathways in
   cancer, JAK/STAT signaling pathways and interestingly in mTOR and TGF-β
   pathways. [84]Fig 5 demonstrates the combined intended schematic KEGG
   signaling pathways. Targets of miR-141 and miR-200a are indicated with
   red stars.

Fig 5. miR-141 and miR-200a targetome are both involved in several important
pathways including TGF-β, mTOR and FOXO, which their partial diagram
collected from KEGG pathway is demonstrated.

   [85]Fig 5
   [86]Open in a new tab

   Red stars mark common targets of miR-141 and miR-200a.

Investigating the expression level of miR-141 and miR-200a’s common targets
defined by molecular pathway enrichment analysis

   Next, to validate our in-silico experiments, several common targets of
   miR-141 and miR-200a such as: FOXO3, SMAD2 and GATA3 (not shown in the
   picture) were chosen for extra analyses of the effect of miR-141 and
   miR-200a on their targets. Interestingly results showed that expression
   level of FOXO3 decreased significantly in relapsing compared to the
   control group (p value = 0.047). Expression level of SMAD2 and GATA3
   displayed similar pattern in which their expression declined
   meaningfully in both relapsing and remitting groups compared to healthy
   group (p value = 0.028 and p value = 0.001 respectively for SMAD2, p
   value = 0.017 and p value = 0.010 respectively for GATA3) ([87]Fig 6).

Fig 6. Target genes of miR-141 and miR-200a specified in-silico were tested
for further analysis and the expression level of target genes of miR-141 and
miR-200a in CD4^+ T cells of relapsing group (n = 20), remitting group (n =
20) and healthy controls (n = 10) were assessed.

   [88]Fig 6
   [89]Open in a new tab

   Results are normalized relative to expression level of reference gene,
   18srRNA (*p < 0.05 and **p < 0.01, non-parametric Mann-Whitney t-test)
   (RP: Relapsing patient, MP: Remitting patient, HV: Healthy volunteer).

Discussion

   As mentioned before, Th17 and Treg cells are involved in pathogenesis
   of MS [[90]4]. Recently, there are growing interest to unravel
   different miRNAs’ roles in various pathways involved in differentiation
   of these cells. However, the main challenge in this scenario is “how”
   and “which” miRNAs participate in differentiation of T helper cells.
   Several studies due to miRNAs in MS were conducted in PBMC, lymphocytes
   and/or other tissues, so far. These studies tried to find and nominate
   miRNAs with utmost importance as biomarkers and/or therapeutic
   solutions. Thus, introducing new miRNAs to the world of autoimmune
   diseases, especially MS and finding their possible role in the course
   of the disease is essential.

   A number of studies explored the correlation between miR-141 and
   miR-200a with different autoimmune diseases such as SLE. Accordingly,
   miR-141 and miR-200a may contribute to other autoimmune diseases with
   the same immune-pathologic mechanism. Hence, we investigated the role
   of miR-141 and miR-200a in pathogenesis if MS.

   In this study, we identified that miR-141 and miR-200a significantly
   elevated in relapsing compared to remitting group of MS and control
   group. In addition our flow cytometry results showed that percentage of
   Th17 cells is higher in relapsing group versus remitting and control
   groups while percentage of Treg cells increased in remitting group.
   Based on our results, we hypothesized that overexpression of miR-141
   and miR-200a in relapsing compared to remitting phase of MS, regulate
   differentiation of Th17 cells while inhibiting differentiation to Treg
   cells.

   Generally, Th17 cells have crucial roles in inducing autoimmunity in
   various autoimmune diseases. On the contrary, Treg cells inhibit
   autoimmunity. Studies on frequency of Th17 and Treg cells in peripheral
   blood in MS also illustrate that number of Th17 cells are found to be
   higher in relapsing phase of MS patients than either remitting or
   healthy controls while Treg cells are found at higher rate in healthy
   controls compared to MS phases [[91]24,[92]25]. As notified, no study
   evaluated the expression level of miR-141 and miR-200a in CD4^+ T cells
   of MS patients so far, but a recent study conducted on B lymphocytes of
   MS patients showed that expression level of miR-200a in B lymphocytes
   of MS patients decreased compared to healthy controls [[93]26] which
   seems rational as they inspected miRNA expression in B lymphocytes
   while the present study is focused on CD4^+ T cells, especially Th17
   and Treg cells. Further studies on miR-141 and miR-200a expression in
   other autoimmune-Th17 related diseases like idiopathic thrombocytopenic
   purpura (ITP) state that expression level of miR-141 was down regulated
   in PBMC of patients [[94]27]. Since PBMC is a cocktail of different
   cells and expression of miRNAs are specific in each cell type, thus
   expression of miRNAs can be highly unpredictable and unreliable in
   PBMC. Another study by Olaru et al. showed up-regulation of miR-141 and
   miR-200a in IBD-dysplasia [[95]28]. Also a study on serum and urinary
   free miRNAs of SLE patients displayed that both miR-141 and miR-200a
   down-regulated in serum and urine of patients [[96]16]. Considering the
   fact that expression and presence of miRNAs in serum and urine are
   different from what is in the cell, we can vindicate our results. While
   writing this manuscript, we found a newly published study explaining
   the overexpression of miR-141 in brain tissues of EAE mice, suggesting
   new perceptions of the role of miRNAs as potential therapeutic agents.
   This is generally consistent with our findings [[97]29].

   In order to further confirm our results, we assessed the expression
   level of IL-17A, IL23R, RORC, TGF-β and FOXP3 in different phases of
   MS. Our analyses showed up-regulation of IL23R, RORC and IL-17A in
   relapsing phase of MS patients.

   IL-17A is one of the most important cytokines of Th17 cells and induce
   inflammatory reactions by stimulating the production of other cytokines
   and chemokine and its increase in serum and CSF of patients has been
   reported [[98]10]. As, IL-23 is required for continuous differentiation
   of Th17 cells, IL23R can mainly be expressed in Th17 cells [[99]30].
   RORC gene transcribes to various isoforms including RORγt (RORC2) which
   is the master transcription factor of Th17 differentiation [[100]31].
   Therefore, our findings approve inclination of Th17 cells in relapsing
   phase of MS patients. Interestingly our data demonstrate that TGF-β
   level elevates in both relapsing and remitting phases of MS. This
   correlates with dual role of TGF-β in both differentiations of Th17 and
   Treg cells. Studies show that high concentrations of TGF-β leads to
   activation of FOXP3, thus differentiation to Treg cell while low
   concentrations of TGF-β leads to activation of STAT3 and RORγt, thus
   differentiation to Th17 cell [[101]32]. Giving this, up-regulation of
   TGF-β in both phases is correlated with Th17/Treg development in
   relapsing and remitting phases respectively. As mentioned earlier,
   FOXP3 is the master transcription factor of Treg cells [[102]8].
   Expression level of FOXP3 displays down regulation in relapsing phase
   compared to control group but no significant increase was found in
   remitting group, presumably due to either the fact that many genes are
   also regulated at the post-transcriptional level [[103]33], hence
   measuring protein level seems to be more accurate, or limited number of
   patients and healthy controls in current study. Consequently, in
   agreement with our flow cytometry results, Th17 cells are increasing in
   relapsing while Treg cells are increasing in remitting phase of MS
   patients which confirm plausible contribution of miR-141 and miR-200a
   in differentiation of T helper cells.

   Our computational analyses revealed that JAK/STAT, TGF-β and mTOR
   signaling pathways, combined with other pathways, are probably among
   the most important pathways affected by miR-141 and miR-200a. Trying to
   nominate multiple important genes which are direct targets of miR-141
   and miR-200a and are also involved in differentiation of T helper
   cells, we selected FOXO3, SMAD2 and GATA3.

   GATA3 is the master regulator of Th2 cells as studies showed that loss
   of GATA3 in peripheral CD4^+ T cells abolish differentiation to Th2
   cells [[104]8]. Moreover, it is now clear that TGF-β induce
   differentiation of both Th17 and Treg cells [[105]32]. TGF-β acts
   through inducing a FOXP3^+ RORγt^+ double positive phenotype in naïve
   CD4^+ T cells as well as inhibiting differentiation to Th1 and Th2
   cells. It has been also stated that SMAD2 and SMAD3, downstream genes
   in TGF-β pathway, are important in differentiation of Th17 cells
   [[106]34]. Lastly, activation of mTORC2 leads to phosphorylation and
   suppression of FOXO3 which is critical for appropriate development of
   Th17 cells. CD4^+ T cells deficient in mTOR do not differentiate into
   either Th1, Th2 or Th17 cells [[107]23,[108]35]. Therefore inhibition
   of FOXO3 together with other cited pathways leads the differentiation
   to Th17 cells. Our analyses also revealed that expression level of
   SMAD2, GATA3 and FOXO3 decline in relapsing phase of MS, concluding
   that their lessened expression level promote Th17 differentiation while
   inhibiting Treg differentiation. Collectively, our findings propose
   that miR-141 and miR-200a may possibly inhibit SMAD2, GATA3 and FOXO3
   through which promote differentiation of Th17 cells and inhibition of
   Treg and/or other T helper subset developmental pathway.

   In conclusion, in current study we indicated that miR-141 and miR-200a
   up-regulate in relapsing compared to remitting phase of MS patients and
   controls. In addition, we showed elevated percentage of Th17,
   simultaneous with reduced level of Treg cells in relapsing phase of MS.
   Thus, we proposed involvement of miR-141 and miR-200a in
   differentiation of T helper cells. Our in-silico analyses further
   approved that miR-141 and miR-200a may involve in pathways of Th17/Treg
   differentiation such as JAK/STAT, TGF-β and mTOR pathways. Finally, we
   also revealed declined expression level of SMAD2, GATA3 and FOXO3, as
   direct targets of miR-141 and miR-200a in relapsing phase of MS. Thus,
   we suggest probable involvement of both miR-141 and miR-200a in
   differentiation of Th17 and Treg cells. Hence, these novel
   Th17-associated miRNAs could be investigated as potential therapeutic
   targets to inhibit MS progression in future. However, additional
   in-vitro and in-vivo experiments are also needed to further confirm
   this phenomenon.

Supporting Information

   S1 Fig. Real time PCR products for miR-141, miR-200a and RNU48 were run
   on poly acrylamide gel and existence of only one band validates the
   specificity of our primers (M:50[bp]).

   (TIF)
   [109]Click here for additional data file.^ (199.9KB, tif)
   S2 Fig. One positive clone from PCR product of each miRNA was selected
   and extracted plasmids were sent for sequencing.

   Sequencing data analyses revealed that miR-141, miR-200a and RNU48
   primers specifically amplified miR-141 (A), miR-200a (B) and RNU48 (C).

   (TIF)
   [110]Click here for additional data file.^ (9.2MB, tif)
   S1 Table. Patient’s clinical appearances.

   (DOC)
   [111]Click here for additional data file.^ (68.5KB, doc)
   S2 Table. In-silico analyses on targets of miR-141 and miR-200a.

   Table A shows predicated and validated targets of miR-141, using
   miRWalk and miRTarBase databases respectively. Similar data are shown
   for miR-200a in Table B. Common targets of both miRs are shown
   separately in Table C. EST profiles of miR-141 and miR-200a which
   contain predicated and validated mRNA targets of miR-141 in thymus and
   lymph node are shown in Tables D and E respectively.

   (XLSX)
   [112]Click here for additional data file.^ (94.7KB, xlsx)

Acknowledgments