MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis

Biochemical and Biophysical Research Communications xxx (2017) 1e8

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis Qing Liu a, *, Qing Gao a, Yu Zhang a, Zhibao Li a, Xiaoxue Mei b a b

Department of Neurology, The Fifth People's Hospital of Jinan, Shandong, China Department of Rehabilitation, The Fifth People's Hospital of Jinan, Shandong, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 September 2017 Accepted 22 September 2017 Available online xxx

Although the exact pathogenesis of multiple sclerosis (MS) remains largely unclear, Th17 cells have been suggested as an essential regulator in the disease induction. Emerging evidence have demonstrated that noncoding RNAs, especially microRNAs (miRs), play a crucial role in modulation of Th17 cell differentiation and autoimmune disease development. Here, we revealed that miR-590 expression was markedly increased in periphery blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) of patients with MS, and positively correlated with the disease severity. Th17 cells were found to express high level of miR-590. We further demonstrated that miR-590 was able to facilitate Th17 differentiation and pathogenicity. Notably, we identified that miR-590 directly targeted Tob1, a known suppressor of Th17 differentiation. The expression level of Tob1 was observed to be significantly decreased in PBMC of patients with MS. Our finding suggest that miR-590 could enhance pathogenic Th17 differentiation in MS and augment inflammation in central nervous system (CNS) through inhibiting Tob1. © 2017 Published by Elsevier Inc.

Keywords: MiR-590 Multiple sclerosis Th17 Tob1

1. Introduction Multiple sclerosis (MS) is an organ-specific chronic demyelinating neurodegenerative disorder of the central nervous system (CNS), leading to damage of the insulating covers of nerve cells in the brain and spinal cord. Patients with MS may present a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems [1]. Although the exact pathogenesis of MS still remains largely unclear, it is accepted that a combination of genetic factors, environmental factors such as viral infection, and dysregulated immune responses jointly contribute to the induction and development of this disease. It has been wildly known that both innate and adaptive immune responses are involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), and CD4þ T-cell-mediated autoimmunity has long been known as one of the most important aspect of MS pathogenesis, especially for the early initiation of disease. Th1 cells, which can produce IFN-g and express T-bet, are originally considered to mediate MS development. Recently, a novel helper CD4þ T cell subtype has been identified and characterized by

* Corresponding author. Department of Neurology, The Fifth People's Hospital of Jinan, 24297 Jingshi Road, Jinan, Shandong, 250022, China. E-mail address: [email protected] (Q. Liu).

secreting interleukin (IL)-17A, IL-17F and IL-21, which was named as Th17 cells. This subtype of effector helper T cells can be induced by stimulation of IL-6, IL-1b, IL-23, and TGF-b, and are strongly suggested to be critical mediators of chronic and autoimmune inflammation [2]. A study using mice with IL-23p19 deficiency demonstrated that lack of IL-23 resulted in resistance to EAE and markedly impaired Th17 cell generation, but IL-12p35/ mice that have a defect in Th1 differentiation were still susceptible to CNS autoimmune inflammation [3]. Notably, administration of anti23p19 monoclonal antibody was capable of ameliorating EAE [4]. RORgt is the lineage-specific transcriptional factor of Th17 and is indispensable for its differentiation, and mice with RORgt deficiency in T cells have attenuated autoimmune disease and downregulated Th17 cell infiltrates in CNS after induction of EAE. Those data strongly suggest that Th17 cells are crucial players in the development of autoimmune diseases. MicroRNAs (miRs) is a group of non-coding RNAs containing about 22 nucleotides, which regulate protein expression at the post-transcriptional level, and emerging studies have documented the importance of miRs in the pathophysiology of autoimmune diseases, including MS. For example, the initiation of TCR signaling could induce miR-31 expression in helper T cells, and miR-31 impaired the differentiation of peripherally derived regulatory T (Treg) cells after antigen stimulation, resulting in an imbalance

https://doi.org/10.1016/j.bbrc.2017.09.123 0006-291X/© 2017 Published by Elsevier Inc.

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123

2

Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8

between pathogenic Th1/Th17 cells and Treg cells, thus augmenting inflammation in the CNS through targeting Gprc5a [5]. General ablation of miRs in T cells by conditional deletion of Dicer that is an indispensable nuclease for the processing of miR led to the spontaneous development of autoimmunity in aged mice [6]. miR-124 was identified as a key regulator of microglia quiescence in the CNS. Its overexpression in bone marrowederived macrophages markedly suppressed the transcription factor CCAAT/enhancerbinding protein-a (C/EBP-a) and its downstream target PU.1, and deactivated these cells, resulting in low expression of CD45, major histocompatibility complex (MHC) class II, and TNF-a [7]. miR-590 has been reported to play a role in modulation of the development of several cancers. It has been found that miR-590 expression was decreased in human colorectal cancer cells and tissues compared to that in normal control cells and tissues, and it could inhibit colorectal cancer angiogenesis and metastasis by regulating nuclear factor 90/vascular endothelial growth factor A axis [8]. Moreover, miR-590 favored the cell proliferation, migration and invasion in Tcell acute lymphoblastic leukaemia by increasing E-cadherin and inhibiting MMP-9 via targeting RB1 [9]. However, whether miR-590 contributes to the regulation of autoimmunity remains unknown. In the current study, we investigated the expression levels of miR-590 in periphery blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) of patients with MS, which was markedly upregulated compared to those in healthy individuals. Mechanistically, miR-590 facilitated the differentiation of naïve CD4þ T cells into a pathogenic Th17 lineage, and we demonstrated Tob1, a member of the tob/btg1 family of anti-proliferative proteins, as a novel target gene of miR-590 [10]. Finally, we found a significantly decrease in the expression of Tob1 in sera and CSF of patients with MS. Our data thus demonstrate that miR-590 could be a potential therapeutic target for MS patients. 2. Materials and methods More detailed information is provided Supplementary Materials and Methods section.

in

the

online

2.1. Subjects All patients and healthy donors were enrolled from Department of Neurology, The Jinan Fifth People's Hospital (Shandong, China), including 42 MS patients during relapsing stage, 36 during remitting stages, and 33 healthy controls. Periphery blood samples were collected in tubes with EDTA anticoagulant after overnight fasting from the same patterns of patients and healthy donors described above. CSF samples were obtained by lumbar puncture performed for diagnosis purposes. The study was approved by the Institutional Review Board for Clinical Research of the Jinan Fifth People's Hospital. 2.2. Lentivirus-mediated CD4þ T cell transduction Lentivirus that encodes pre-miR-590 (LV-miR-590) and the control vector (LV-miR-ctrl) were purchased from Genechem (Shanghai, China). CD4þ T cells were transduced with LV-miR-590 and LV-scramble, respectively, according to the manufacturer's protocol. These CD4þ T cells were then cultured and stimulated with plate-bound anti-CD3 (5 mg/ml) and anti-CD28 (2 mg/ml). On the third day after transduction, CD4þ T cells were harvested for further analysis for mRNA levels for IL-17A, RORC, IL-17F, CXCL3, CSF2, IL-23R, AHR, IL-10 and MAF using qRT-PCT.

the predicted seed sequence in the 30 -UTR of Tob1, as well as the accordingly mutant vectors in which three random nucleotide mutants were introduced into the seed sequences. The Renilla luciferase activity was then determined as described previously [11]. Briefly, we cultured HEK 293T cells in complete DMEM supplemented with 10% FBS, 10 mM HEPES, 100 U/ml penicillin/ streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, and 50 mM 2-ME at 37  C in 5% CO2/95% air under humidified conditions. We seeded 293T cells in a 24-well plate (2  106/well) and transfected them with recombinant dual-luciferase vectors alone, vectors plus LV-miR-590, and vectors plus LV-miR-ctrl, respectively, using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Luciferase assays were performed 24 h later using the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA). The Renilla and firefly luciferase signals were measured on the Veritas Microplate Luminometer (Promega, Fitchburg, WI, USA). 2.4. Statistical analysis Data are presented as mean ± SEM. We performed statistical comparisons using an unpaired 2-tailed Student's t-test, One-way ANOVA, or Pearson's correlation analysis. A value of P < 0.05 was considered significant. 3. Results 3.1. MiR-590 expression is significantly increased in patients with MS To determine the expression level of miR-590 in patients with MS, we obtained periphery blood from 42 patients with MS in the relapsing stage (relapse-MS), 36 with MS who were in the remitting stage (remit-MS), and 33 healthy individuals. We next collected PBMC and performed qRT-PCR to examine miR-590 expression in those samples. miR-590 was highly up-regulated in PBMC from patients with relapse-MS compared with that in patients with remit-MS and healthy individuals (Fig. 1A). It has been welldocumented that neurofilament light chain (NF-L) is a molecular marker of MS, expression of which is associated with the disease severity [12e14], and we found that PBMC expression of miR-590 was positively associated with NF-L expression in sera of patients with relapse-MS (Fig. 1B). Moreover, we found that PBMC expression of miR-590 positively correlated with serum C-reaction protein (CRP) and erythrocyte sedimentation rate (ESR) levels in patients with relapse-MS (Fig. 1C and D), both of which have been well-known to be increased in MS patients compared to those in healthy controls. Since MS mainly affects the CNS, we next aimed to reveal the local expression pattern of miR-590 in CNS of patients. We collected CSF samples from those subjects described in Fig. 1A, and observed that patients with relapse-MS displayed significantly increased miR-590 expression in CSF than patients with remit-MS and healthy controls (Fig. 1E), which was consistent with the observation in Fig. 1A. Similarly, miR-590 was positively correlated with NF-L expression in CSF, as well as CXCL13 and CHI3L1 (Fig. 1FeH), both of which were also identified as the biomarkers to evaluate the disease severity and treatment efficacy in MS [12], Taken together, these evidence suggest that miR-590 is significantly up-regulated in MS patients, and might play a pathogenic role in the progression of the disease. 3.2. Th17 cells express high levels of miR-590

2.3. Luciferase reporter assay for target validation Dual-luciferase reporter vectors were constructed containing

In order to investigate the pathogenesis underlying the upregulation of miR-590 in MS, we first sought to identify the

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123

Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8

3

Fig. 1. Patients with multiple sclerosis (MS) exhibits increased miR-590 expression. (A) Quantitative real-time reverse transcription (qRT)-PCR analysis of miR-590 expression in periphery blood mononuclear cell (PMBC) of patients with MS who were in the relapsing stage (relapse-MS, n ¼ 42), patients with MS who were in the remitting stage (remit-MS, n ¼ 36), and healthy donors (HC, n ¼ 33). ***P < 0.001. A correlation between miR-590 expression and serum neurofilament light chain (NF-L) expression (B), CRP expression (C), and ESR (D) in patients with relapse-MS was evaluated using Spearman's Rank Correlation Analysis. (E) Lumbar cerebrospinal fluid (CSF) samples were collected from subjects described in Fig. 1A, and miR-590 expression was examined using qRT-PCR. ***P < 0.001. Correlation between miR-590 expression and NF-L (F), CXCL13 (G), and CHI3L1 (H) expression in CSF of patients with relapse-MS were analyzed using Spearman's Rank Correlation Analysis, respectively. miR-590 expression was normalized to endogenous U6.

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123

4

Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8

cellular source of miR-590 expression. To this end, CD4þ, CD8þ T cells, B cells, and neutrophils were isolated from periphery blood of 15 healthy donors. Notably, we found that CD4þ T cells were responsible for high expression of miR-590, while its expression was much lower in CD8þ T cells and B cells, even undetectable in neutrophils (Fig. 2A). Given the function varies among the lineages of CD4þ T cells, we next aimed to investigate the expression pattern of miR-590 in different subtypes of CD4þ T cells. To do so, we differentiated naïve CD4þ T cells under different polarizing conditions, and as showed in Fig. 2B, miR-590 was found to be expressed at the highest level in Th17 cells, implicating that miR-590 might play a role in driving Th17 differentiation. We further revealed that miR-590 expression closely positively correlated with IL-17A and RORC expression in both periphery blood and CSF of patients with relapse-MS (Fig. 2CeF). These data implicate that miR-590 is probably involved in the differentiation and function of Th17 cells.

3.3. MiR-590 promotes pathogenic Th17 cell differentiation To investigate whether miR-590 contribute to Th17 differentiation, we transduced periphery naïve CD4þ T cells with LV-miR-590 and LV-miR-ctrl, respectively. LV-miR-590 transduction significantly increased the expression level of miR-590 in CD4þ T cells (online Supplementary Fig. S1). The mRNA expression of IL-17A and RORC (Fig. 3A and B) as well as IL-17A protein expression (Fig. 3D and E) was remarkably up-regulated after transduction with LVmiR-590 compared with cells with LV-miR-ctrl or cultured alone in medium 5 days after stimulation with anti-CD3/CD28. We also found that STAT3 phosphorylation was highly enhanced by miR-590 overexpression (Fig. 3C), and thus we suggested that miR-590 might facilitate Th17 differentiation via activating STAT3. To further verify the role of miR-590 in IL-17A expression, we transduced naïve CD4þ T cells as described above, and cultured these cells in Th17 polarizing

Fig. 2. Th17 cells express high levels of miR-590. (A) Analysis of miR-590 expression in different subsets of immune cells. Periphery blood CD4þ T cells, CD8þ T cells, B cells, neutrophils were collected from 15 healthy donors, and miR-590 expression was determined by qRT-PCR. **P < 0.01, ***P < 0.001. (B) Examination of miR-590 expression in different subsets of helper T cells, induced from periphery naïve CD4þ T cells. miR-590 expression in each subset was determined by qRT-PCR. **P < 0.01, ***P < 0.001. There was a significant positive correlation between miR-590 expression and protein levels of IL-17A in serum (C) and CSF (D) samples of patients with relapse-MS. Furthermore, miR-590 expression significantly correlated with RORC mRNA levels in PBMC (E) and CSF (F) of patients with relapse-MS. miR-590 expression was normalized to endogenous U6 and RORC mRNA was normalized to GAPDH.

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123

Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8

5

Fig. 3. MiR-590 promotes Th17 cell differentiation. Peripheral naive CD4þ T cells were transduced with lentiviral vectors encoding pre-miR-590 (LV-miR-590) and control lentivirus (LV-miR-ctrl), respectively, and then stimulated in vitro with plate-bound anti-CD3 and anti-CD28. After 5 days, cells were harvested and analyzed for the levels of IL-17A (A) and RORC (B) by qRT-PCR. (C) Western blot was performed to assess STAT3 phosphorylation. (D) Intracellular expression of IL-17A was analyzed by flow cytometry. (E) Percentage of IL17Aþ CD4þ T cells was shown in bar chart. (FeI) Naïve CD4þ T cells were transduced as described above and cultured in Th17-skewing condition for 5 days. IL-17A (F) and RORC (G) expression was examined by qRT-PCR. (H) Intracellular expression of IL-17A was analyzed by flow cytometry. (I) Percentage of IL-17Aþ CD4þ T cells was shown in bar chart. **P < 0.01, ***P < 0.001 compared with that in cells harboring LV-miR-ctrl or cultured in medium alone. miR-590 expression was normalized to endogenous U6 and mRNA expression was normalized to GAPDH.

condition under stimulation with anti-CD3/CD28. Consistently, overexpression of miR-590 significantly promoted IL-17A and RORC mRNA as well as IL-17A protein expression under Th17-skewing condition (Fig. 3FeI). Since emerging evidence have indicated that Th17 cells can be divided into pathogenic and non-pathogenic subsets [15,16], we therefore sought to investigate whether miR590 could affect Th17 pathogenicity. As demonstrated in online Supplementary Figs. S2AeC, CXCL3, CSF2, and IL-23R, which are associated with Th17 pathogenicity, were significantly up-regulated after LV-miR-590 transduction into CD4þ T cells with Th17-skewing

culture condition. In note, enforced expression of miR-590 markedly decreased the transcript levels of AHR, IL-10, and MAF, which are known as nonpathogenic molecules (online Supplementary Figs. S2DeF). Collectively, these findings strongly suggest that miR-590 is able to promote pathogenic Th17 cell differentiation. 3.4. MiR-590 targets Tob1 that is significantly decreased in patients with MS In an attempt to further clarify the mechanism by which miR-

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123

6

Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8

590 modulates the pathogenesis of MS, we sought to determine the downstream targets of miR-590 that modulate CNS inflammation. We then utilized three computer-based miR target-detection programs (Miranda, targerscan, and miRDB) to predict the potential target genes of miR-590. As shown in Fig. 4A, we found Tob1 had a

potential binding site for miR-590. According to a previous report, Tob1 played a critical role in the activation of encephalitogenic T cells in CNS autoimmunity, especially Th17 cells [10], so we focused on exploring whether Tob1 was indeed a target gene of miR-590. Luciferase reporter assay was performed and co-transduction of

Fig. 4. MiR-590 targets Tob1. (A) The predicted binding sites of miR-590 in the Tob1 30 -UTR. (B) Luciferase assay and lentivirus-mediated transduction was performed as indicated and the luciferase activity of the Tob1 or mutant Tob1 (Tob1 mut) 30 -UTR reporter in 293T cells was assessed. *P < 0.05. (C) The inhibitory effect of miR-590 on Tob1 expression in CD4þ T cells was confirmed by transduction of periphery naïve CD4þ T cells isolated from healthy donors and MS patients with LV-miR-590 or LV-miR-ctrl. Tob1 expression was analyzed by qRT-PCR. *P < 0.05. (D) Tob1 expression in PMBC obtained from subjects described in Fig. 1A was analyzed by qRT-PCR. ***P < 0.001. (E) An inverse correlation between miR-590 and Tob1 expression in PBMC from patients with MS were observed. Peripheral naive CD4þ T cells were transduced with lentiviral vectors encoding Tob1 siRNA (LV-Tob1 siRNA) and control siRNA (LV-control siRNA), respectively, and cultured in Th17-skewing condition for 5 days. The expression of CXCL3 (F), CSF2 (G), IL-23R (H), AHR (I), IL-10 (J), and MAF (K) was analyzed by qRT-PCR and compared with that in cells with LV-control siRNA or cultured in medium alone. *P < 0.05, **P < 0.01, ***P < 0.001. miR-590 expression was normalized to endogenous U6 and mRNA expression was normalized to GAPDH.

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123

Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8

the LV-miR-590 highly inhibited the activity of Renilla luciferase which contained the seed sequence in the 30 -UTR of Tob1 (Fig. 4B), indicating that miR-590 specifically suppressed Tob1 expression. We next transduced CD4þ T cells with LV-miR-590 and LV-miR-ctrl, respectively, and the expression level of Tob1 mRNA was significantly suppressed by enforced miR-590 expression, when compared to that in cells harboring LV-miR-ctrl (Fig. 4C). To investigate whether Tob1 is indeed a functional target of miR-590 in MS, we then investigated Tob1 expression in PMBC of patients with relapseMS, which was remarkably decreased, compared to that in remit-MS patients or healthy controls (Fig. 4D). As expected, an inverse association was observed between miR-590 and Tob1 expression in PBMC from patients with relapse-MS (Fig. 4E). To further verify that miR-590 promoted Th17 pathogenicity via inhibiting Tob1, we transduced CD4þ T cells from healthy donors with LV-Tob1 siRNA, which specifically inhibits the expression of Tob1 (online Supplementary Fig. S3A), and cultured these cells under Th17 polarizing condition. As shown in online Supplementary Figs. S3B and C, knockdown of Tob1 markedly increased IL-17A expression. Moreover, LV-Tob1 siRNA significantly increased the expression of CXCL3, CSF2, and IL-23R, while decreased the transcript levels of AHR, IL-10, and MAF (Fig. 4FeK). Taken together, our data suggest that miR-590 promotes pathogenic Th17 differentiation through targeting Tob1 in MS and aggravates the disease. 4. Discussion The Th1/Th2 paradigm of CD4þ T cell subsets has been revised when Th17 cells characterized by producing IL-17 were identified as a novel T helper cell lineage. Th17 cells are potent to protect the host against certain extracellular pathogens, particularly gramnegative bacteria and fungi and play as an essential driver of organ specific autoimmunity. Given IL-17 was discovered to be upregulated in human autoimmune diseases like MS, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD) as well as in animal models of autoimmunity, researchers have paid much attention to defining the role of Th17 cells in the pathogenesis of autoimmune inflammation [17,18]. Over the past decade, the importance of Th17 cells in the pathogenic process of organ-specific autoimmune disease has been identified in different animal models. IL-23p19 knockout mice are resistant to EAE and knockout of IL-17 in mice result in delayed and attenuated CNS inflammation [19]. In addition, local chemokine expression in the brain and the subsequent EAE development is largely abrogated after introduction of an IL-17-blocking antibody to mice immunized with a myelin antigen [4,20]. In the current study, we demonstrated that miR-590 play a role in promoting pathogenic Th17 cell differentiation through inhibition of Tob1 in the pathogenesis of MS. Although Th17 cells have been recognized as key mediators in the pathogenesis of MS, the exact mechanism still remains unclear. Many molecules and pathways and that could be involved in the process have been suggested. Noncoding RNAs, especially miRs, have drawn much attention to their role in regulating immune responses. Emerging reports have added to mounting evidence linking changes in miR expression to Th17 differentiation and the development of autoimmunity. For example, miR-301a has been reported to be significantly up-regulated in IBD and MS, and it facilitated Th17 cell differentiation both in the gut and CNS [11,21,22]. In the EAE model, miR-301a contributed to the development of the Th17 subset via targeting the IL-6/23eSTAT3 pathway, and manipulation of miR-301a expression with specific antagomir leaded to significant changes in the severity of EAE [21]. miR-21 knockout mice which displayed impaired Th17 differentiation were resistant to EAE. Furthermore, miR-21 promoted Th17 responses through inhibiting SMAD-7, a negative regulator of TGF-b

7

signaling. Intravenous administration of WT mice with an antisense oligonucleotide to inhibit miR-21 significantly reduced the clinical severity of EAE, accompanied by a decrease in the number of Th17 cells [23]. miR-155 functioned to enhance the development of effector T cells including Th1 and Th17 cell subsets. Furthermore, miR-155/ mice were highly resistant to EAE induced by MOG35e55 and exhibited defective Th17 cell development during the disease [24]. In this study, we found miR-590 to be significantly up-regulated in patients with relapse-MS and intimately correlated with several inflammatory markers. miR-590 expression was particularly pronounced in Th17 cells, and most significantly, miR590 overexpression increased the pathogenicity of Th17 cells by upregulating several inflammation-associated molecules, such as CXCL3, CSF2, and IL-23R. Our data demonstrated that Tob1 is a target gene of miR-590. Tob1 is a member of the Tob/BTG antiproliferative (APRO) family of proteins and it acts as a crucial suppressor in tumor development. Tob1, together with other members of this family (TOB2, BTG1, PC3/TIS21/BTG2, ANA/BTG3 and PC3/PC3K) share a common N-terminal domain, which composed of ~120 residues. This domain is considered to presumably confer an antiproliferative effect on these genes. Tob1 was identified to be constitutively expressed in unstimulated periphery blood T lymphocytes, but antigen-specific and unspecific stimulation powerfully decreased its expression. Of note, the proliferation and activation of T cells were remarkably restored by Tob1 inhibition [25]. A recent report revealed that Tob1 expression was significantly decreased in CD4þ T cells of patients with an initial CNS demyelinating event, correlated with high risk for progression to MS. In addition, Tob1/ mice developed much severer EAE after MOG35e55 administration and the infiltrating of inflammatory cells including CD4þ T cell counts was highly increased, when compared with wild type mice. In particular, the number of myelin-reactive Th17 cells was significantly increased, whereas Treg cell expansion was strongly inhibited [10]. Here, we found that miR-590 expression was inversely correlated with Tob1 in patients with relapse-MS, and miR-590 might facilitate pathogenic Th17 cell differentiation through targeting Tob1. In conclusion, our data has demonstrated, for the first time, that miR-590 is a pathogenic Th17 subset-associated miR and significantly up-regulated in both PBMC and CSF of patients with MS. Tob1, a known suppressor of Th17 commitment, is a putative target of miR-590. Our study has shed a new light on understanding the pathogenic role of miR-590 in the pathogenesis of MS and it might represent a crucial target for potential therapeutic intervention. Funding This work was funded by grants from the National Natural Science Foundation of China (81565291). Conflicts of interest The authors have declared that no conflict of interest exists. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2017.09.123. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2017.09.123.

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123

8

Q. Liu et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8

References [1] A.G. Baxter, The origin and application of experimental autoimmune encephalomyelitis, Nat. Rev. Immunol. 7 (2007) 904e912. [2] T. Korn, E. Bettelli, M. Oukka, et al., IL-17 and Th17 cells, Annu. Rev. Immunol. 27 (2009) 485e517. [3] D.J. Cua, J. Sherlock, Y. Chen, et al., Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain, Nature 421 (2003) 744e748. [4] C.L. Langrish, Y. Chen, W.M. Blumenschein, et al., IL-23 drives a pathogenic T cell population that induces autoimmune inflammation, J. Exp. Med. 201 (2005) 233e240. [5] L. Zhang, F. Ke, Z. Liu, et al., MicroRNA-31 negatively regulates peripherally derived regulatory T-cell generation by repressing retinoic acid-inducible protein 3, Nat. Commun. 6 (2015) 7639. [6] C. Xiao, K. Rajewsky, MicroRNA control in the immune system: basic principles, Cell 136 (2009) 26e36. [7] E.D. Ponomarev, T. Veremeyko, N. Barteneva, et al., MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway, Nat. Med. 17 (2011) 64e70. [8] Q. Zhou, Y. Zhu, X. Wei, et al., MiR-590-5p inhibits colorectal cancer angiogenesis and metastasis by regulating nuclear factor 90/vascular endothelial growth factor A axis, Cell Death Dis. 7 (2016) e2413. [9] M.H. Miao, X.Q. Ji, H. Zhang, et al., miR-590 promotes cell proliferation and invasion in T-cell acute lymphoblastic leukaemia by inhibiting RB1, Oncotarget 7 (2016) 39527e39534. [10] U. Schulze-Topphoff, S. Casazza, M. Varrin-Doyer, et al., Tob1 plays a critical role in the activation of encephalitogenic T cells in CNS autoimmunity, J. Exp. Med. 210 (2013) 1301e1309. [11] C. He, T. Yu, Y. Shi, et al., MicroRNA 301A promotes intestinal inflammation and colitis-associated cancer development by inhibiting BTG1, Gastroenterology 152 (2017) 1434e1448 e1415. [12] L. Novakova, M. Axelsson, M. Khademi, et al., Cerebrospinal fluid biomarkers of inflammation and degeneration as measures of fingolimod efficacy in multiple sclerosis, Mult. Scler. 23 (2017) 62e71. [13] J. Kuhle, C. Barro, G. Disanto, et al., Serum neurofilament light chain in early

[14]

[15] [16] [17] [18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

relapsing remitting MS is increased and correlates with CSF levels and with MRI measures of disease severity, Mult. Scler. 22 (2016) 1550e1559. S. Modvig, M. Degn, H. Roed, et al., Cerebrospinal fluid levels of chitinase 3like 1 and neurofilament light chain predict multiple sclerosis development and disability after optic neuritis, Mult. Scler. 21 (2015) 1761e1770. Y. Lee, A. Awasthi, N. Yosef, et al., Induction and molecular signature of pathogenic TH17 cells, Nat. Immunol. 13 (2012) 991e999. Z. Hasan, S.I. Koizumi, D. Sasaki, et al., JunB is essential for IL-23-dependent pathogenicity of Th17 cells, Nat. Commun. 8 (2017) 15628. S.L. Gaffen, R. Jain, A.V. Garg, et al., The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing, Nat. Rev. Immunol. 14 (2014) 585e600. L. Steinman, A brief history of T(H)17, the first major revision in the T(H)1/ T(H)2 hypothesis of T cell-mediated tissue damage, Nat. Med. 13 (2007) 139e145. Y. Komiyama, S. Nakae, T. Matsuki, et al., IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis, J. Immunol. 177 (2006) 566e573. H.H. Hofstetter, S.M. Ibrahim, D. Koczan, et al., Therapeutic efficacy of IL-17 neutralization in murine experimental autoimmune encephalomyelitis, Cell Immunol. 237 (2005) 123e130. M.P. Mycko, M. Cichalewska, A. Machlanska, et al., MicroRNA-301a regulation of a T-helper 17 immune response controls autoimmune demyelination, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) E1248eE1257. C. He, Y. Shi, R. Wu, et al., miR-301a promotes intestinal mucosal inflammation through induction of IL-17A and TNF-alpha in IBD, Gut 65 (2016) 1938e1950. G. Murugaiyan, A.P. da Cunha, A.K. Ajay, et al., MicroRNA-21 promotes Th17 differentiation and mediates experimental autoimmune encephalomyelitis, J. Clin. Invest. 125 (2015) 1069e1080. R.M. O'Connell, D. Kahn, W.S. Gibson, et al., MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development, Immunity 33 (2010) 607e619. D. Tzachanis, G.J. Freeman, N. Hirano, et al., Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells, Nat. Immunol. 2 (2001) 1174e1182.

Please cite this article in press as: Q. Liu, et al., MicroRNA-590 promotes pathogenic Th17 cell differentiation through targeting Tob1 and is associated with multiple sclerosis, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.09.123