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Th2 via targeting RUNX3 in psoriasis
Immunology Letters 166 (2015) 55–62
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
MicroRNA-138 regulates the balance of Th1/Th2 via targeting RUNX3 in psoriasis Dandan Fu a , Wenfa Yu b , Min Li a , Huimin Wang b , Dong Liu a , Xiangfeng Song c , Zhanguo Li a , Zhongwei Tian a,∗ a
Department of Dermatology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453000, Henan, China Department of Otolaryngological, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453000, Henan, China c Department of Immunology, Xinxiang Medical University, Weihui 453000, Henan, China b
a r t i c l e
i n f o
Article history: Received 24 November 2014 Received in revised form 13 May 2015 Accepted 25 May 2015 Available online 1 June 2015 Keywords: Psoriasis RUNX3 microRNA-138 CD4+ T cells
a b s t r a c t Psoriasis is a common chronic inflammatory and T cell-meditated autoimmune skin disease. A recent study found that Runt-related transcription factor 3 (RUNX3) is a susceptibility gene for psoriasis; however, its biological role in the disease has not been studied. RUNX3 was predicted to be the target gene of microRNA-138 (miR-138). The current research was designed to delineate the mechanism of miR138 in regulating psoriasis via targeting RUNX3. In this study, we found that the expression of RUNX3 is increased significantly while the expression of miR-138 decreased significantly in CD4+ T cells from psoriasis patients. Moreover, the luciferase report confirmed the targeting reaction between miR-138 and RUNX3. After transfection with the miR-138 inhibitor into CD4+ T cells from healthy controls, we found that the inhibition of miR-138 increases RUNX3 expression and increased the ratio of Th1/Th2. Furthermore, the miR-138 mimic was transfected into CD4+ T cells from psoriasis patients. The results showed that the overexpression of miR-138 inhibits RUNX3 expression and decreased the ratio of Th1/Th2 in CD4+ T cells. Taken together, our study suggests that increased miR-138 regulates the balance of Th1/Th2 through inhibiting RUNX3 expression in psoriasis, providing a potential therapeutic target for psoriasis. © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Psoriasis is a chronic inflammatory skin disease with a disturbed balance in the interplay of immune cells and keratinocytes, which affects 2∼3% of the population [1,2]. Psoriasis is thought to be a T cell-mediated disease of autoimmune origin [3]. Disease manifestation is orchestrated by pro-inflammatory CD4 positive (CD4+ ) T helper (Th) cells. It has been reported that psoriasis is a Th1/Th17-induced inflammatory autoimmune disease and is mainly mediated by Th1 cells [4,5]. It has also been reported that interleukin (IL)-4-producing Th2 cells play a protective role in psoriasis, which is closely associated with the deviation of Th1 into Th2
Abbreviations: Th, T helper; IL, interleukin; RUNX3, Runt-related transcription factor 3; microRNA-138, miR-138; miRNAs, MicroRNAs; MERs, miRNA recognition elements; 3 -UTR3 , untranslated region; mRNAs, messenger RNAs; PASI, psoriasis area severity index. ∗ Corresponding author at: Department of Dermatology, The First Affiliated Hospital of Xinxiang Medical University, NO. 88 Healthy road, Weihui 453000, Henan, China. Tel.: +86 0373 4402634. E-mail address: [email protected] (Z. Tian).
responses [6]. Recent studies showed that genetics play an important role in the development of psoriasis and more than 30 loci have been reported to be associated with predisposition to the disease to date [7–9]. Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene that participates in the regulation of cell proliferation and apoptosis. RUNX3 is commonly expressed in bone marrow, spleen, thymus, peripheral blood, spinal cord cells, B cells and T cells [10]. The changes in RUNX3 protein levels or functions are associated with various human diseases; for example, the inactivation of RUNX3 is associated not only with gastric cancer but also with various other epithelial cancers [11]. It has been reported that Runx3-deficient mice spontaneously develop two immunological abnormalities: colitis and airway hypersensitivity [12,13]. In addition, the deletion of Runx3 in T cells resulted in the spontaneous development of asthma-like features in targeted mice (elevated IgE, IgA, IgG1 and infiltration of lymphocytes and eosinophils in the lung) [14]. These studies suggest that the regulation of RUNX3 participates in many immune pathologies. A recent study reported that RUNX3 expression was significantly increased in psoriasis patients compared with normal controls, and indicates that RUNX3
http://dx.doi.org/10.1016/j.imlet.2015.05.014 0165-2478/© 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
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is a susceptibility gene for psoriasis [15]. Moreover, it is reported that RUNX3 plays an important role in the differentiation of T cells [16], affecting the balance of Th1/Th2; thus, RUNX3 may affect the development and occurrence of psoriasis. Formerly, researchers have focused on the coding region when studying the mechanism of one specific gene. Nowadays, the role of noncoding regions has become increasingly more important for investigating the function of genes. MicroRNAs (miRNAs) are endogenous, noncoding RNAs consisting of 21–23 nucleotides [17]. MiRNAs regulate gene expression through binding to miRNA recognition elements (MERs) located in the 3 untranslated region (3 -UTR) of target messenger RNAs (mRNAs), leading to their translational repression or degradation [18]. It has been estimated that ∼60% of all protein-coding genes in humans are regulated by miRNAs which participate in the regulation of almost every cellular process investigated to date [19]. Multiple studies have shown that miRNAs play a crucial role in the pathogenesis of psoriasis. For example, miR-203 [20], mir-125b [21], miR-99a [22] and miR424 [23] regulate keratinocyte proliferation and differentiation. In addition, up-regulation of miR-21 suppressed T cell apoptosis in psoriasis [24]. These studies reveal an important role for miRNAs in the biology of psoriasis. In this study, we identified a function for miR-138 in the context of psoriasis. We found that the expression of miR-138 is significantly decreased in CD4+ T cells of psoriasis patients, leading to overexpression of the target gene RUNX3, which regulates the balance of Th1/Th2. Taken together, our results suggest that miR-138 may provide novel insight into the dysregulation of psoriasis via targeting RUNX3. 2. Materials and methods 2.1. Patients Forty patients (22 males and 18 females) with psoriasis and 35 healthy subjects (18 males and 17 females) were consecutively included in this study. Patients with psoriasis were recruited from the dermatological department of the First Affiliated Hospital, Xinxiang Medical University. All patients were categorized according to disease severity using the psoriasis area severity index (PASI). Healthy subjects were recruited from students at the Xinxiang Medical University. The study was approved by the Ethics Committee of our institution (code 2014055). Informed consent was signed by the participants. 2.2. Specimen collection Fasting venous peripheral blood samples were drawn from each patient and control subject, and were preserved with heparin. CD4+ T cells were isolated using a positive selection magnetic column (Miltenyi, Bergisch Gladbach, Germany). The blood CD4+ T lymphocyte absolute counts were measured by flow cytometry. CD4+ T cell purity typically exceeded 90% according to the protocols, and were cultured in human T cell culture medium (OpTmizerTM CTSTM T-Cell Expansion SFM, Gibco, Rockville, MD, USA). 2.3. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from CD4+ T cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The purity and concentration of total RNA were determined by an Ultraviolet Spectrophotometer (Eppendorf, German). cDNAs were synthesized using the One Step PrimeScript miRNA cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). mRNA was analyzed by real-time PCR using primers synthesized by the Shanghai Sangon Biological Engineering and Technology Service (Shanghai, China). Briefly, 20 l reactions
containing 50 ng of total RNA, 10 l of 2× SYBR Green PCR Master Mix, 6.25 U of AMV reverse transcriptase, 10 U of RNase inhibitor and 0.1 mM of primers were subjected to one cycle of 95 ◦ C for 10 min and then 40 cycles of 95 ◦ C for 15 s, 56 ◦ C for 30 s and 72 ◦ C for 45 s. miR-138 expression was normalized to the U6 RNA. Gene mRNA expression was normalized to ˇ-actin. Data were analyzed by the Ct method and expressed as fold-changes.
2.4. Construction of the luciferase reporter vector A 300 bp sequence from the 3 -UTR of RUNX3 containing a putative miR-138 binding site was amplified by PCR using the cDNA of human CD4+ T cells as a template. The sequence for the mutation within the miR-138 binding site was amplified by the point mutation method using the KOD-Plus mutagenesis Kit (TOYOBO, Osaka, Japan) according to the protocols provided. After cloning into the pGEM-T vector (Promega, Madison, Wisconsin, USA), the PCR product was purified and inserted into a pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, Austin, Texas, USA) using SpeI and HindIII (Takara Biotechnology, Dalian, China). The recombinant plasmids were confirmed by DNA sequencing.
2.5. Transfection and activity assay of the luciferase reporter vector MOLT-4 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS). Using LipofectamineTM 2000, the recombinant plasmids and 100 nM Hsa-miR-138 mimics, miR-138 mimic control, Hsa-miR-138 inhibitor, inhibitor control (Ambion, Austin, Texas, USA) were transfected into MOLT-4 cells respectively for 48 h. The firefly luciferase and renilla luciferase activity were detected by a fluorescence detector (Promega, Madison, Wisconsin, USA); relative luciferase activity was normalized to renilla luciferase activity for each transfected well. The experiment was replicated three times for data calculate.
2.6. CD4+ T cell transfection CD4+ T cells were transfected with Hsa-miR-138 mimic, HsamiR-138 inhibitor, miR-138 mimic control or miR-138 inhibitor control according to the instructions provided with the Lipofectamine RNAi MA× reagent. After 48 h of transfection, cells were collected for qRT-PCR to detect the mRNA expression of RUNX3 and other further analysis.
2.7. Western blotting Total proteins from the CD4+ T cells were extracted using RIPA lysis buffer (Beyotime, Nantong, China) and quantified with the BCA assay (Pierce, Rockford, IL). Equal amounts of protein were loaded and separated by 10% or 15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). After transferring onto nitrocellulose membranes and blocking in Tris-buffered saline-Tween buffer containing 5% nonfat dry milk for 30 min, the target proteins were incubated overnight at 4 ◦ C with RUNX3 or -actin rabbit anti-human antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were incubated with the corresponding goat anti-rabbit secondary antibodies for 1 h at room temperature. A fluorescent Western blotting detection system was used. The band density of each gene was normalized to the corresponding density of -actin.
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Fig. 1. Expression of miR-138 and RUNX3 in CD4+ T cells from psoriasis patients and healthy controls. (A) The expression levels of miR-138 were measured by quantitative real-time PCR (qRT-PCR); (B) the mRNA expression levels of RUNX3 were measured by qRT-PCR; (C) the protein expression levels of RUNX3 were measured by Western blotting. Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to ˇ-actin; (D) the correlation between miR-138 expression levels and RUNX3 protein expression levels in CD4+ T cells from psoriasis patients. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus control group.
2.8. Enzyme-linked immunosorbent assay (ELISA)
3. Result
The content of cytokine concentrations was measured by the ELISA method using IL-2, IL-4, IL-10, and IFN-␥ ELISA kits (R&D, Minneapolis, MN, USA), according to the manufacturer’s protocols. Optical density (OD value) at the wavelength of 450 nm was measured using a Microplate Reader (BioTek, Winooski, VT, USA) after the experiment. The concentration of samples was calculated according to the corresponding OD value and concentration of the standard substance.
3.1. Increased expression of RUNX3 and decreased expression of miR-138 in psoriasis CD4+ T cells
2.9. Flow cytometry The percentage of Th1 and Th2 in CD4+ cells was detected by flow cytometry analysis. Briefly, cells were incubated with Ionomycin, phorbol ester (PMA) and monensin (all purchased from Sigma St. Louis, MO, USA) fro 4 h at 37 ◦ C. Then stined with FITClabeled anti-CD4, PE-labeled anti-IFN␥ and FITC-labeled anti-CD4, PE-labeled anti-IL4 (BD, San Diego, USA) respectively for 30 min at room temperature. Th1 cells were identified as CD4+ IFN␥+ , and Th2 cells were identified as CD4+ IL4+ . The results were calculated by CELLQuest software.
2.10. Statistical analysis All data were processed with SPSS13.0. Measurement data were presented as mean ± standard deviation. Differences between groups were compared by standard deviation followed by independent-samples T test. Repeated analysis of variance (ANOVA) was used for comparison between groups. A difference was considered significant at p < 0.05.
CD4+ T cells were isolated from venous peripheral blood samples from each patient and control subject. The expression of mature miR-138 was measured in healthy subjects (n = 35) and psoriasis patients (n = 40) using qRT-PCR. The results showed that the expression of miR-138 was significantly decreased in CD4+ T cells from psoriasis patients as compared with healthy controls (Fig. 1A). Next, the expression of RUNX3 was examined by qRT-PCR and Western blot analysis. The results showed that both the mRNA and protein levels of RUNX3 were significantly elevated in CD4+ T cells from psoriasis patients compared with healthy controls (Fig. 1B and C). In addition, there was a strong negative correlation between the expression level of miR-138 and the protein expression level of RUNX3 in CD4+ T cells from psoriasis patients (Fig. 1D).
3.2. Increased level of Th1 related cytokines and decreased level of Th2 related cytokines in psoriasis CD4+ T cells To investigate the role of Th1 and Th2 in psoriasis, we evaluated the level of Th1 and Th2 related cytokines in psoriasis CD4+ T cells. The content of Th1 cell-related cytokines (IL-2, IFN-␥) and Th2 cell-related cytokines (IL-4, IL-10) were measured using the ELISA method (Fig. 2A). The results showed that the level of IL-2 and IFN-␥ were significantly increased, while the level of IL-4, IL-10 were significantly decreased in psoriasis patients compared with healthy controls. Moreover, we detected the expression level of Th1-related transcription factor T-box expressed in T cells (T-bet) and Th2-related transcription factor GATA binding protein 3 (GATA3) using qRT-PCR (Fig. 2B). The results showed that the
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Fig. 2. Expression level of Th1 and Th2 related cytokines in CD4+ T cells from psoriasis patients and healthy controls. (A) The protein level of IL-2, IFN-␥, IL-4 and IL-10 were measured by the ELISA method; (B) the mRNA expression level of T-bet and GATA3 were measured by qRT-PCR. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus control group.
expression level of T-bet was significantly increased while the expression level of GATA3 was significantly decreased in psoriasis patients compared with healthy controls, which were consistent with the former results. 3.3. The prediction and identification of RUNX3 targeted miRNA The bioinformatic software programs miRbase, RegRNA and RNAhybird were used to predict the RUNX3 targeted miRNA. According to the results, the potential binding target sites of miR138 were found in the 3 -UTR of RUNX3 gene (Fig. 3A). The luciferase reporter vectors of RUNX3-wt and RUNX3-mut, which contained the wild type and mutant type of potential binding sequence in 3 UTR of RUNX3, respectively, were constructed and co-transfected with miR-138 mimics or miR-138 mimic controls into MOLT-4 cells to identify the target region of miR-138 and RUNX3. After 48 h of
transfection, the results showed that the luciferase activity in the RUNX3-wt with miR-138 mimics group was significantly reduced compared with the other three groups. However, there was no significant difference in luciferase activity among co-transfection groups of RUNX3-wt with the miR-138 mimic control, RUNX3mut with miR-138 mimics and RUNX3-mut with the miR-138 mimic control (Fig. 3B). In addition, the luciferase reporter vectors of RUNX3-wt and RUNX3-mut were co-transfected with miR-138 inhibitors or inhibitor controls to further confirm the targeting reaction between miR-138 and RUNX3. The results showed that the luciferase activity in the RUNX3-wt with miR-138 inhibitor cotransfection group was increased significantly compared with the other three groups. Moreover, there was no significant difference in luciferase activity among co-transfection groups of RUNX3-wt with the miR-138 inhibitor control, RUNX3-mut with miR-138 inhibitor and RUNX3-mut with the miR-138 inhibitor control (Fig. 3C).
Fig. 3. Identification of the targeting reaction between RUNX3 and miR-138. The target gene was predicted by bioinformatic software programs, identified by luciferase activity report. (A) The wild miR-138 binding sequence and the mutant miR-138 binding sequence in the 3 -UTR of the RUNX3 gene; (B) relative luciferase activity in MOLT-4 cells co-transfected with miR-138 mimic or mimic control together with luciferase reporter constructs containing either wild type (wt) or mutant type (mut) RUNX3 3 -UTR; (C) relative luciferase activity in MOLT-4 cells co-transfected with miR-138 inhibitor or inhibitor control together with luciferase reporter vector wt-RUNX3 or mut-RUNX3. Luciferase activities were represented as firefly luciferase normalized to renilla luciferase. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus other three groups.
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Fig. 4. Inhibition of miR-138 increases RUNX3 expression and regulates the cytokines related to Th1 and Th2 in CD4+ T cells from healthy controls. The CD4+ T cells from healthy controls were transfected with miR-138 inhibitor or inhibitor control. (A) Expression levels of miR-138 were measured by qRT-PCR; (B and E) the mRNA expression levels of RUNX3, T-bet and GATA3 were measured by qRT-PCR; (C) the protein expression levels of RUNX3 were measured by Western blotting. Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to ˇ-actin; (D) the protein level of IL-2, IFN-␥, IL-4 and IL-10 were measured by the ELISA method. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus inhibitor control.
3.4. Inhibition of miR-138 increases RUNX3 expression and regulate the cytokines related with Th1 and Th2 in CD4+ T cells from healthy controls The miR-138 inhibitor and inhibitor control were transfected into CD4+ T cells from healthy controls to investigate the effect of miR-138 inhibition in normal CD4+ T cells. After 48 h of transfection, the expression of miR-138 was detected using RT-PCR. The results showed that miR-138 expression was decreased significantly in CD4+ T cells transfected with the miR-138 inhibitor compared with the inhibitor control (Fig. 4A). The mRNA and protein levels of RUNX3 were detected using qRT-PCR and Western blotting. The results showed that the mRNA level of RUNX3 was significantly increased in CD4+ T cells transfected with the miR-138
inhibitor compared with the inhibitor control (Fig. 4B), which is consistent with the result of Western blotting (Fig. 4C). Next, the content of Th1 cell-related cytokines (IL-2, IFN␥) and Th2 cell-related cytokines (IL-4, IL-10) were measured using the ELISA method. Moreover, expression of the Th1related transcription factor T-bet and the Th2-related transcription factor GATA3 were measured by RT-PCR. The results showed that IL-2 and IFN-␥ were up-regulated and IL-4 and IL-10 were down-regulated significantly in CD4+ T cells transfected with the miR-138 inhibitor compared with the inhibitor control (Fig. 4D). Similarly, the expression of T-bet was increased and the expression of GATA3 was decreased in the inhibitor transfection of CD4+ T cells compared to the inhibitor control (Fig. 4E).
Fig. 5. Overexpression of miR-138 inhibits RUNX3 expression and regulates the cytokines related to Th1 and Th2 in CD4+ T cells from psoriasis patients. The CD4+ T cells from psoriasis patients were transfected with the miR-138 mimic or mimic control. (A) Expression levels of miR-138 were measured by qRT-PCR; (B and E) the mRNA expression levels of RUNX3, T-bet and GATA3 were measured by qRT-PCR; (C) the protein expression levels of RUNX3 were measured by Western blotting. Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to ˇ-actin; (D) the protein levels of IL-2, IFN-␥, IL-4 and IL-10 were measured by ELISA. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus mimic control.
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Fig. 6. Overexpression of miR-138 regulates the deviation of Th1/Th2 in CD4+ T cells from psoriasis patients. The percentages of IFN-␥+ CD4+ T cells and IL-4+ CD4+ T cells in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control were measured by flow cytometry. (A and B) Flow cytometric analysis of Th1 in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control. The percentages of cells in Q2 region represent the percentage of CD4+ IFN-␥+ T cells. (C and D) Flow cytometric analysis of Th2 in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control. The percentage of cells in Q2 was the percentage of CD4+ IL-4+ T cells. (E) The frequency of Th1 and Th2 cells from psoriasis patients transfected with mimic or mimic control. (F) The frequency ratio of Th1 and Th2 in CD4+ T cells from psoriasis patients transfected with mimic or mimic control. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus mimic control.
3.5. Overexpression of miR-138 inhibits RUNX3 expression and regulates the cytokines related to Th1 and Th2 in CD4+ T cells from psoriasis patients The miR-138 mimic and mimic control were transfected into CD4+ T cells from psoriasis patients to detect whether the overexpression of miR-138 could reverse the immune dysfunction. miR-138 expression was detected after 48 h of transfection using qRT-PCR. The results showed that miR-138 expression was significantly increased in CD4+ T cells transfected with miR-138 mimic compared with the mimic control (Fig. 5A). In addition, the mRNA and protein expression of RUNX3 was decreased significantly with miR-138 mimic transfection (Fig. 5B and C). Furthermore, the content of Th1 and Th2 cell-related cytokines was also detected, as well as the expression of Th1 related transcription factor T-bet and Th2-related transcription factor GATA3. The ELISA results showed that IL-2 and IFN-␥ were down-regulated and IL-4 and IL-10 were up-regulated significantly in CD4+ T cells transfected with miR-138 mimic compared with the mimic control (Fig. 5D). The qRT-PCR results showed that the expression of T-bet
was decreased and the expression of GATA3 was increased in the mimic-transfected CD4+ T cells when compared with the mimic control (Fig. 5E).
3.6. Overexpression of miR-138 regulates the balance of Th1/Th2 in CD4+ T cells from psoriasis patients The percentages of IFN-␥+ CD4+ T cells and IL-4+ CD4+ T cells in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control were detected by flow cytometry. The results showed that the percentage of IFN-␥+ CD4+ T cells in the mimic group was decreased compared to those of the mimic control group (Fig. 6A and B). Moreover, the percentage of IL-4+ CD4+ T cells in the mimic group was increased compared to the mimic control group (Fig. 6C and D). Next, the ratio of Th1/Th2 in CD4+ T cells transfected with mimic or mimic control was calculated. The results showed that the ratio of Th1/Th2 in the mimic group was significantly lower than in the mimic control group (Fig. 6F).
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4. Discussion Psoriasis is one of the most common inflammatory and T cellmediated autoimmune diseases [1]. Tsoi et al. reported that RUNX3 is a susceptibility gene for psoriasis [15]. Consistently, we found that the expression of RUNX3 is significantly increased in CD4+ T cells from psoriasis patients in the present study. To investigate the role of RUNX3 in psoriasis, the noncoding region, 3 -UTR of RUNX3 was studied in this research. MiRNAs are noncoding RNAs that can suppress the expression of protein-coding genes by binding to the target sequence at the 3 -UTR of the target gene [17]. It has been reported that miRNAs play an important role in various diseases, including psoriasis [25]. After miRNA prediction and filtration using bioinformatic software programs, several miRNA were predicted to have target reaction with RUNX3. Among these miRNAs, we selected miR-138 to be the targeted miRNA fro RUNX3, which may be involved in the pathogenesis of psoriasis. In a previous study, miR-138 was observed to be downregulated in psoriasis [26]. It has been reported that the down-regulation of miR-138 is involved in several biological processes in different diseases. For example, miR-138 could induce cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma [27], could suppress ovarian cancer cell invasion and metastasis by targeting HIF-1a and SOX4 [28], and could inhibit glioblastoma cell proliferation in vitro and tumorigenicity in vivo via inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop [29]. Thus, we chose miR138 to be the targeted miRNA for RUNX3. In our study, the results showed miR-138 was decreased significantly in psoriasis patients compared with healthy controls, which was consistent with former study [26]. Furthermore, we detected a strong negative correlation between the expression level of miR-138 and the protein expression level of RUNX3 in CD4+ T cells from psoriasis patients. To verify the targeting reaction between miR-138 and RUNX3, the luciferase reporter vectors of wild type and mutant RUNX3 were constructed. The results showed that the overexpression of miR-138 inhibits luciferase expression when cells were transfected with wt-RUNX3 luciferase reporter system, but not in mut-RUNX3 groups. Moreover, the inhibition of miR-138 increases luciferase activity in wt-RUNX3 transfection group compared with mut-RUNX3 groups. These results demonstrate that RUNX3 is a target gene for miR-138. As has been previously reported, CD4+ T cells can differentiate down to Th1, Th2, Th17 and other type of T cells depending on cytokines, environmental signals and the nature of the encountered stimulus itself [30]. Ghoreschi et al. reported that the balance of Th1 and Th2 is associated with the development of psoriasis [31]. In addition, RUNX3 plays an important role in the differentiation of T cells and affects the balance of Th1/Th2 [16]. Thus, to investigate the role of miR-138 in T cell differentiation via targeting RUNX3, we detected the effect of miR-138 inhibition on expression of RUNX3 and deviation of Th1/Th2 in CD4+ T cells from healthy controls. The results showed that RUNX3 is significantly increased due to the inhibition of miR-138, which is similar to the physiological feature in psoriasis. Djuretic et al. reported that the expression of RUNX3 is related to transcription factor T-beta in the process of Th1 cells differentiation [32]. Moreover, RUNX3 augments Th1 cells and reduce Th2 cells by interacting with and attenuating GATA3 [33]. In the present study, we detected the expression level of Tbet and GATA3, as well as Th1-related cytokines (IL-2, IFN-␥) and Th2-related cytokines (IL-4, IL-10) in miR-138 inhibitor-transfected healthy CD4+ T cells. The results showed that the inhibition of miR-138 induces Th1-related cytokines and increased the expression of T-bet, whereas this inhibits Th2-related cytokines and also decreased the expression of GATA3. Taken together, these results suggest that the inhibition of miR-138 induce the deviation of Th1/Th2 by increasing RUNX3 expression, which may lead to the development of psoriasis.
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Recent studies have discovered several miRNAs that are potential therapeutic targets for psoriasis. Guinea-Viniefar et al. demonstrated that the inhibition of miR-21 ameliorated disease pathology in patient-derived psoriatic skin xenotransplants in mice and in a psoriasis-like mouse model [34]. Zhao et al. reported that the down-regulation of miR-210 improves immune dysfunction in CD4+ T cells of psoriasis patients [35]. In our study, we detected the effect of miR-138 overexpression in CD4+ T cells from psoriasis patients. The results showed that RUNX3 expression was inhibited due to the overexpression of miR-138. Moreover, the balance of Th1/Th2 was broken and shifted to overexpress of Th2-related cytokines. Furthermore, the result of flow cytometric analysis showed that the ratio of Th1/Th2 in CD4+ T cells decreased significantly after transfection with the miR-138 mimic. These results suggest that the overexpression of miR-138 regulates the balance of Th1/Th2, leading to immune dysfunction improvement via inhibition of the expression of RUNX3. In conclusion, our results demonstrated that the downregulation of miR-138 affects the expression of RUNX3, leading to an imbalance of Th1/Th2, which is involved in the pathogenesis of psoriasis. This study provides an important clue to help to elucidate the pathogenesis of psoriasis and implicates miR-138 as a potential therapeutic target for psoriasis.
Acknowledgments The study was supported by Science and Technology Research Key Project of The Education Department Henan Province (Grant NO. 13A320852), Science and Technology Research Plan in Xinxiang City (Grant No. ZG13022) and Health and Science Innovative Talents Project in Henan Province (Grant No. 201004159).
References [1] F.O. Nestle, D.H. Kaplan, J. Barker, Psoriasis N. Engl. J. Med. 361 (2009) 496–509. [2] M.A. Lowes, A.M. Bowcock, J.G. Krueger, Pathogenesis and therapy of psoriasis, Nature 445 (2007) 866–873. [3] C.E. Griffiths, J.N. Barker, Pathogenesis and clinical features of psoriasis, The Lancet 370 (2007) 263–271. [4] E. Lee, W.L. Trepicchio, J.L. Oestreicher, D. Pittman, F. Wang, F. Chamian, et al., Increased expression of interleukin 23 p19 and p40 in lesional skin of patients with psoriasis vulgaris, J. Exp. Med. 199 (2004) 125–130. [5] K. Ghoreschi, M. Rocken, Molecular and cellular basis for designing gene vaccines against inflammatory autoimmune disease, Trends Mol. Med. 9 (2003) 331–338. [6] K. Ghoreschi, P. Thomas, S. Breit, M. Dugas, R. Mailhammer, W. van Eden, et al., Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease, Nat. Med. 9 (2003) 40–46. [7] H. Tang, X. Jin, Y. Li, H. Jiang, X. Tang, X. Yang, et al., A large-scale screen for coding variants predisposing to psoriasis, Nat. Genet. 46 (2014) 45–50. [8] Garber K. Genetics, Deep exploration, Nature 492 (2012) S56–S57. [9] R.P. Nair, K.C. Duffin, C. Helms, J. Ding, P.E. Stuart, D. Goldgar, et al., Genomewide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways, Nat. Genet. 41 (2009) 199–204. [10] R. Yagi, I.S. Junttila, G. Wei, J.F. Urban Jr., K. Zhao, W.E. Paul, et al., The transcription factor GATA3 actively represses RUNX3 protein-regulated production of interferon-gamma, Immunity 32 (2010) 507–517. [11] K. Blyth, E.R. Cameron, J.C. Neil, The RUNX genes: gain or loss of function in cancer, Nat. Rev. Cancer 5 (2005) 376–387. [12] O. Brenner, D. Levanon, V. Negreanu, O. Golubkov, O. Fainaru, E. Woolf, et al., Loss of Runx3 function in leukocytes is associated with spontaneously developed colitis and gastric mucosal hyperplasia, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 16016–16021. [13] O. Fainaru, E. Woolf, J. Lotem, M. Yarmus, O. Brenner, D. Goldenberg, et al., Runx3 regulates mouse TGF-beta-mediated dendritic cell function and its absence results in airway inflammation, EMBO J. 23 (2004) 969–979. [14] Y. Naoe, R. Setoguchi, K. Akiyama, S. Muroi, M. Kuroda, F. Hatam, et al., Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer, J. Exp. Med. 204 (2007) 1749–1755. [15] L.C. Tsoi, S.L. Spain, J. Knight, E. Ellinghaus, P.E. Stuart, F. Capon, et al., Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity, Nat. Genet. 44 (2012) 1341–1348. [16] I.M. Djuretic, F. Cruz-Guilloty, A. Rao, Regulation of gene expression in peripheral T cells by Runx transcription factors, Adv. Immunol. 104 (2009) 1–23.
62
D. Fu et al. / Immunology Letters 166 (2015) 55–62
[17] V. Ambros, R.C. Lee, A. Lavanway, P.T. Williams, D. Jewell, MicroRNAs and other tiny endogenous RNAs in C. elegans, Curr. Biol. 13 (2003) 807–818. [18] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2009) 215–233. [19] H. Siomi, M.C. Siomi, Posttranscriptional regulation of microRNA biogenesis in animals, Mol Cell. 38 (3) (2010) 323–332. [20] E. Sonkoly, T. Wei, P.C. Janson, A. Saaf, L. Lundeberg, M. Tengvall-Linder, et al., MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoS One 2 (2007) e610. [21] N. Xu, P. Brodin, T. Wei, F. Meisgen, L. Eidsmo, N. Nagy, et al., MiR-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2, J. Invest. Dermatol. 131 (2011) 1521–1529. [22] G. Lerman, C. Avivi, C. Mardoukh, A. Barzilai, A. Tessone, B. Gradus, et al., MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R, PLoS One 6 (2011) e20916. [23] A. Ichihara, M. Jinnin, K. Yamane, A. Fujisawa, K. Sakai, S. Masuguchi, et al., microRNA-mediated keratinocyte hyperproliferation in psoriasis vulgaris, Br. J. Dermatol. 165 (2011) 1003–1010. [24] F. Meisgen, N. Xu, T. Wei, P.C. Janson, S. Obad, O. Broom, et al., MiR-21 is upregulated in psoriasis and suppresses T cell apoptosis, Exp. Dermatol. 21 (2012) 312–314. [25] T. Tomankova, M. Petrek, J. Gallo, E. Kriegova, MicroRNAs: emerging regulators of immune-mediated diseases, Scand. J. Immunol. 78 (2011) 129–141. [26] J.R. Zibert, M.B. Lovendorf, T. Litman, J. Olsen, B. Kaczkowski, L. Skov, MicroRNAs and potential target interactions in psoriasis, J. Dermatol. Sci. 58 (2010) 177–185.
[27] Y. Jin, D. Chen, R.J. Cabay, A. Wang, D.L. Crowe, X. Zhou, Role of microRNA-138 as a potential tumor suppressor in head and neck squamous cell carcinoma, Int. Rev. Cell Mol. Biol. 303 (2013) 357–385. [28] Y.M. Yeh, C.M. Chuang, K.C. Chao, L.H. Wang, MicroRNA-138 suppresses ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1alpha, Int. J. Cancer 133 (2013) 867–878. [29] S. Qiu, D. Huang, D. Yin, F. Li, X. Li, H.F. Kung, et al., Suppression of tumorigenicity by microRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme, Biochim. Biophys. Acta 1832 (2013) 1697–1707. [30] H. Kared, X. Camous, A. Larbi, T cells and their cytokines in persistent stimulation of the immune system, Curr. Opin. Immunol. 29 (2014) 79–85. [31] K. Ghoreschi, C. Weigert, M. Rocken, Immunopathogenesis and role of T cells in psoriasis, Clin. Dermatol. 25 (2007) 574–580. [32] I.M. Djuretic, D. Levanon, V. Negreanu, Y. Groner, A. Rao, K.M. Ansel, Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells, Nat. Immunol. 8 (2007) 145–153. [33] K. Kohu, H. Ohmori, W.F. Wong, D. Onda, T. Wakoh, S. Kon, et al., The Runx3 transcription factor augments Th1 and down-modulates Th2 phenotypes by interacting with and attenuating GATA3, J. Immunol. 183 (2009) 7817–7824. [34] J. Guinea-Viniegra, M. Jimenez, H.B. Schonthaler, R. Navarro, Y. Delgado, M.J. Concha-Garzon, et al., Targeting miR-21 to treat psoriasis, Sci. Transl. Med. 6 (2014) 225re221. [35] M. Zhao, L.T. Wang, G.P. Liang, P. Zhang, X.J. Deng, Q. Tang, et al., Up-regulation of microRNA-210 induces immune dysfunction via targeting FOXP3 in CD4(+) T cells of psoriasis vulgaris, Clin. Immunol. 150 (2014) 22–30.
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
MicroRNA-138 regulates the balance of Th1/Th2 via targeting RUNX3 in psoriasis Dandan Fu a , Wenfa Yu b , Min Li a , Huimin Wang b , Dong Liu a , Xiangfeng Song c , Zhanguo Li a , Zhongwei Tian a,∗ a
Department of Dermatology, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453000, Henan, China Department of Otolaryngological, The First Affiliated Hospital of Xinxiang Medical University, Weihui 453000, Henan, China c Department of Immunology, Xinxiang Medical University, Weihui 453000, Henan, China b
a r t i c l e
i n f o
Article history: Received 24 November 2014 Received in revised form 13 May 2015 Accepted 25 May 2015 Available online 1 June 2015 Keywords: Psoriasis RUNX3 microRNA-138 CD4+ T cells
a b s t r a c t Psoriasis is a common chronic inflammatory and T cell-meditated autoimmune skin disease. A recent study found that Runt-related transcription factor 3 (RUNX3) is a susceptibility gene for psoriasis; however, its biological role in the disease has not been studied. RUNX3 was predicted to be the target gene of microRNA-138 (miR-138). The current research was designed to delineate the mechanism of miR138 in regulating psoriasis via targeting RUNX3. In this study, we found that the expression of RUNX3 is increased significantly while the expression of miR-138 decreased significantly in CD4+ T cells from psoriasis patients. Moreover, the luciferase report confirmed the targeting reaction between miR-138 and RUNX3. After transfection with the miR-138 inhibitor into CD4+ T cells from healthy controls, we found that the inhibition of miR-138 increases RUNX3 expression and increased the ratio of Th1/Th2. Furthermore, the miR-138 mimic was transfected into CD4+ T cells from psoriasis patients. The results showed that the overexpression of miR-138 inhibits RUNX3 expression and decreased the ratio of Th1/Th2 in CD4+ T cells. Taken together, our study suggests that increased miR-138 regulates the balance of Th1/Th2 through inhibiting RUNX3 expression in psoriasis, providing a potential therapeutic target for psoriasis. © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Psoriasis is a chronic inflammatory skin disease with a disturbed balance in the interplay of immune cells and keratinocytes, which affects 2∼3% of the population [1,2]. Psoriasis is thought to be a T cell-mediated disease of autoimmune origin [3]. Disease manifestation is orchestrated by pro-inflammatory CD4 positive (CD4+ ) T helper (Th) cells. It has been reported that psoriasis is a Th1/Th17-induced inflammatory autoimmune disease and is mainly mediated by Th1 cells [4,5]. It has also been reported that interleukin (IL)-4-producing Th2 cells play a protective role in psoriasis, which is closely associated with the deviation of Th1 into Th2
Abbreviations: Th, T helper; IL, interleukin; RUNX3, Runt-related transcription factor 3; microRNA-138, miR-138; miRNAs, MicroRNAs; MERs, miRNA recognition elements; 3 -UTR3 , untranslated region; mRNAs, messenger RNAs; PASI, psoriasis area severity index. ∗ Corresponding author at: Department of Dermatology, The First Affiliated Hospital of Xinxiang Medical University, NO. 88 Healthy road, Weihui 453000, Henan, China. Tel.: +86 0373 4402634. E-mail address: [email protected] (Z. Tian).
responses [6]. Recent studies showed that genetics play an important role in the development of psoriasis and more than 30 loci have been reported to be associated with predisposition to the disease to date [7–9]. Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene that participates in the regulation of cell proliferation and apoptosis. RUNX3 is commonly expressed in bone marrow, spleen, thymus, peripheral blood, spinal cord cells, B cells and T cells [10]. The changes in RUNX3 protein levels or functions are associated with various human diseases; for example, the inactivation of RUNX3 is associated not only with gastric cancer but also with various other epithelial cancers [11]. It has been reported that Runx3-deficient mice spontaneously develop two immunological abnormalities: colitis and airway hypersensitivity [12,13]. In addition, the deletion of Runx3 in T cells resulted in the spontaneous development of asthma-like features in targeted mice (elevated IgE, IgA, IgG1 and infiltration of lymphocytes and eosinophils in the lung) [14]. These studies suggest that the regulation of RUNX3 participates in many immune pathologies. A recent study reported that RUNX3 expression was significantly increased in psoriasis patients compared with normal controls, and indicates that RUNX3
http://dx.doi.org/10.1016/j.imlet.2015.05.014 0165-2478/© 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.
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is a susceptibility gene for psoriasis [15]. Moreover, it is reported that RUNX3 plays an important role in the differentiation of T cells [16], affecting the balance of Th1/Th2; thus, RUNX3 may affect the development and occurrence of psoriasis. Formerly, researchers have focused on the coding region when studying the mechanism of one specific gene. Nowadays, the role of noncoding regions has become increasingly more important for investigating the function of genes. MicroRNAs (miRNAs) are endogenous, noncoding RNAs consisting of 21–23 nucleotides [17]. MiRNAs regulate gene expression through binding to miRNA recognition elements (MERs) located in the 3 untranslated region (3 -UTR) of target messenger RNAs (mRNAs), leading to their translational repression or degradation [18]. It has been estimated that ∼60% of all protein-coding genes in humans are regulated by miRNAs which participate in the regulation of almost every cellular process investigated to date [19]. Multiple studies have shown that miRNAs play a crucial role in the pathogenesis of psoriasis. For example, miR-203 [20], mir-125b [21], miR-99a [22] and miR424 [23] regulate keratinocyte proliferation and differentiation. In addition, up-regulation of miR-21 suppressed T cell apoptosis in psoriasis [24]. These studies reveal an important role for miRNAs in the biology of psoriasis. In this study, we identified a function for miR-138 in the context of psoriasis. We found that the expression of miR-138 is significantly decreased in CD4+ T cells of psoriasis patients, leading to overexpression of the target gene RUNX3, which regulates the balance of Th1/Th2. Taken together, our results suggest that miR-138 may provide novel insight into the dysregulation of psoriasis via targeting RUNX3. 2. Materials and methods 2.1. Patients Forty patients (22 males and 18 females) with psoriasis and 35 healthy subjects (18 males and 17 females) were consecutively included in this study. Patients with psoriasis were recruited from the dermatological department of the First Affiliated Hospital, Xinxiang Medical University. All patients were categorized according to disease severity using the psoriasis area severity index (PASI). Healthy subjects were recruited from students at the Xinxiang Medical University. The study was approved by the Ethics Committee of our institution (code 2014055). Informed consent was signed by the participants. 2.2. Specimen collection Fasting venous peripheral blood samples were drawn from each patient and control subject, and were preserved with heparin. CD4+ T cells were isolated using a positive selection magnetic column (Miltenyi, Bergisch Gladbach, Germany). The blood CD4+ T lymphocyte absolute counts were measured by flow cytometry. CD4+ T cell purity typically exceeded 90% according to the protocols, and were cultured in human T cell culture medium (OpTmizerTM CTSTM T-Cell Expansion SFM, Gibco, Rockville, MD, USA). 2.3. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from CD4+ T cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The purity and concentration of total RNA were determined by an Ultraviolet Spectrophotometer (Eppendorf, German). cDNAs were synthesized using the One Step PrimeScript miRNA cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). mRNA was analyzed by real-time PCR using primers synthesized by the Shanghai Sangon Biological Engineering and Technology Service (Shanghai, China). Briefly, 20 l reactions
containing 50 ng of total RNA, 10 l of 2× SYBR Green PCR Master Mix, 6.25 U of AMV reverse transcriptase, 10 U of RNase inhibitor and 0.1 mM of primers were subjected to one cycle of 95 ◦ C for 10 min and then 40 cycles of 95 ◦ C for 15 s, 56 ◦ C for 30 s and 72 ◦ C for 45 s. miR-138 expression was normalized to the U6 RNA. Gene mRNA expression was normalized to ˇ-actin. Data were analyzed by the Ct method and expressed as fold-changes.
2.4. Construction of the luciferase reporter vector A 300 bp sequence from the 3 -UTR of RUNX3 containing a putative miR-138 binding site was amplified by PCR using the cDNA of human CD4+ T cells as a template. The sequence for the mutation within the miR-138 binding site was amplified by the point mutation method using the KOD-Plus mutagenesis Kit (TOYOBO, Osaka, Japan) according to the protocols provided. After cloning into the pGEM-T vector (Promega, Madison, Wisconsin, USA), the PCR product was purified and inserted into a pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, Austin, Texas, USA) using SpeI and HindIII (Takara Biotechnology, Dalian, China). The recombinant plasmids were confirmed by DNA sequencing.
2.5. Transfection and activity assay of the luciferase reporter vector MOLT-4 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS). Using LipofectamineTM 2000, the recombinant plasmids and 100 nM Hsa-miR-138 mimics, miR-138 mimic control, Hsa-miR-138 inhibitor, inhibitor control (Ambion, Austin, Texas, USA) were transfected into MOLT-4 cells respectively for 48 h. The firefly luciferase and renilla luciferase activity were detected by a fluorescence detector (Promega, Madison, Wisconsin, USA); relative luciferase activity was normalized to renilla luciferase activity for each transfected well. The experiment was replicated three times for data calculate.
2.6. CD4+ T cell transfection CD4+ T cells were transfected with Hsa-miR-138 mimic, HsamiR-138 inhibitor, miR-138 mimic control or miR-138 inhibitor control according to the instructions provided with the Lipofectamine RNAi MA× reagent. After 48 h of transfection, cells were collected for qRT-PCR to detect the mRNA expression of RUNX3 and other further analysis.
2.7. Western blotting Total proteins from the CD4+ T cells were extracted using RIPA lysis buffer (Beyotime, Nantong, China) and quantified with the BCA assay (Pierce, Rockford, IL). Equal amounts of protein were loaded and separated by 10% or 15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). After transferring onto nitrocellulose membranes and blocking in Tris-buffered saline-Tween buffer containing 5% nonfat dry milk for 30 min, the target proteins were incubated overnight at 4 ◦ C with RUNX3 or -actin rabbit anti-human antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were incubated with the corresponding goat anti-rabbit secondary antibodies for 1 h at room temperature. A fluorescent Western blotting detection system was used. The band density of each gene was normalized to the corresponding density of -actin.
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Fig. 1. Expression of miR-138 and RUNX3 in CD4+ T cells from psoriasis patients and healthy controls. (A) The expression levels of miR-138 were measured by quantitative real-time PCR (qRT-PCR); (B) the mRNA expression levels of RUNX3 were measured by qRT-PCR; (C) the protein expression levels of RUNX3 were measured by Western blotting. Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to ˇ-actin; (D) the correlation between miR-138 expression levels and RUNX3 protein expression levels in CD4+ T cells from psoriasis patients. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus control group.
2.8. Enzyme-linked immunosorbent assay (ELISA)
3. Result
The content of cytokine concentrations was measured by the ELISA method using IL-2, IL-4, IL-10, and IFN-␥ ELISA kits (R&D, Minneapolis, MN, USA), according to the manufacturer’s protocols. Optical density (OD value) at the wavelength of 450 nm was measured using a Microplate Reader (BioTek, Winooski, VT, USA) after the experiment. The concentration of samples was calculated according to the corresponding OD value and concentration of the standard substance.
3.1. Increased expression of RUNX3 and decreased expression of miR-138 in psoriasis CD4+ T cells
2.9. Flow cytometry The percentage of Th1 and Th2 in CD4+ cells was detected by flow cytometry analysis. Briefly, cells were incubated with Ionomycin, phorbol ester (PMA) and monensin (all purchased from Sigma St. Louis, MO, USA) fro 4 h at 37 ◦ C. Then stined with FITClabeled anti-CD4, PE-labeled anti-IFN␥ and FITC-labeled anti-CD4, PE-labeled anti-IL4 (BD, San Diego, USA) respectively for 30 min at room temperature. Th1 cells were identified as CD4+ IFN␥+ , and Th2 cells were identified as CD4+ IL4+ . The results were calculated by CELLQuest software.
2.10. Statistical analysis All data were processed with SPSS13.0. Measurement data were presented as mean ± standard deviation. Differences between groups were compared by standard deviation followed by independent-samples T test. Repeated analysis of variance (ANOVA) was used for comparison between groups. A difference was considered significant at p < 0.05.
CD4+ T cells were isolated from venous peripheral blood samples from each patient and control subject. The expression of mature miR-138 was measured in healthy subjects (n = 35) and psoriasis patients (n = 40) using qRT-PCR. The results showed that the expression of miR-138 was significantly decreased in CD4+ T cells from psoriasis patients as compared with healthy controls (Fig. 1A). Next, the expression of RUNX3 was examined by qRT-PCR and Western blot analysis. The results showed that both the mRNA and protein levels of RUNX3 were significantly elevated in CD4+ T cells from psoriasis patients compared with healthy controls (Fig. 1B and C). In addition, there was a strong negative correlation between the expression level of miR-138 and the protein expression level of RUNX3 in CD4+ T cells from psoriasis patients (Fig. 1D).
3.2. Increased level of Th1 related cytokines and decreased level of Th2 related cytokines in psoriasis CD4+ T cells To investigate the role of Th1 and Th2 in psoriasis, we evaluated the level of Th1 and Th2 related cytokines in psoriasis CD4+ T cells. The content of Th1 cell-related cytokines (IL-2, IFN-␥) and Th2 cell-related cytokines (IL-4, IL-10) were measured using the ELISA method (Fig. 2A). The results showed that the level of IL-2 and IFN-␥ were significantly increased, while the level of IL-4, IL-10 were significantly decreased in psoriasis patients compared with healthy controls. Moreover, we detected the expression level of Th1-related transcription factor T-box expressed in T cells (T-bet) and Th2-related transcription factor GATA binding protein 3 (GATA3) using qRT-PCR (Fig. 2B). The results showed that the
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Fig. 2. Expression level of Th1 and Th2 related cytokines in CD4+ T cells from psoriasis patients and healthy controls. (A) The protein level of IL-2, IFN-␥, IL-4 and IL-10 were measured by the ELISA method; (B) the mRNA expression level of T-bet and GATA3 were measured by qRT-PCR. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus control group.
expression level of T-bet was significantly increased while the expression level of GATA3 was significantly decreased in psoriasis patients compared with healthy controls, which were consistent with the former results. 3.3. The prediction and identification of RUNX3 targeted miRNA The bioinformatic software programs miRbase, RegRNA and RNAhybird were used to predict the RUNX3 targeted miRNA. According to the results, the potential binding target sites of miR138 were found in the 3 -UTR of RUNX3 gene (Fig. 3A). The luciferase reporter vectors of RUNX3-wt and RUNX3-mut, which contained the wild type and mutant type of potential binding sequence in 3 UTR of RUNX3, respectively, were constructed and co-transfected with miR-138 mimics or miR-138 mimic controls into MOLT-4 cells to identify the target region of miR-138 and RUNX3. After 48 h of
transfection, the results showed that the luciferase activity in the RUNX3-wt with miR-138 mimics group was significantly reduced compared with the other three groups. However, there was no significant difference in luciferase activity among co-transfection groups of RUNX3-wt with the miR-138 mimic control, RUNX3mut with miR-138 mimics and RUNX3-mut with the miR-138 mimic control (Fig. 3B). In addition, the luciferase reporter vectors of RUNX3-wt and RUNX3-mut were co-transfected with miR-138 inhibitors or inhibitor controls to further confirm the targeting reaction between miR-138 and RUNX3. The results showed that the luciferase activity in the RUNX3-wt with miR-138 inhibitor cotransfection group was increased significantly compared with the other three groups. Moreover, there was no significant difference in luciferase activity among co-transfection groups of RUNX3-wt with the miR-138 inhibitor control, RUNX3-mut with miR-138 inhibitor and RUNX3-mut with the miR-138 inhibitor control (Fig. 3C).
Fig. 3. Identification of the targeting reaction between RUNX3 and miR-138. The target gene was predicted by bioinformatic software programs, identified by luciferase activity report. (A) The wild miR-138 binding sequence and the mutant miR-138 binding sequence in the 3 -UTR of the RUNX3 gene; (B) relative luciferase activity in MOLT-4 cells co-transfected with miR-138 mimic or mimic control together with luciferase reporter constructs containing either wild type (wt) or mutant type (mut) RUNX3 3 -UTR; (C) relative luciferase activity in MOLT-4 cells co-transfected with miR-138 inhibitor or inhibitor control together with luciferase reporter vector wt-RUNX3 or mut-RUNX3. Luciferase activities were represented as firefly luciferase normalized to renilla luciferase. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus other three groups.
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Fig. 4. Inhibition of miR-138 increases RUNX3 expression and regulates the cytokines related to Th1 and Th2 in CD4+ T cells from healthy controls. The CD4+ T cells from healthy controls were transfected with miR-138 inhibitor or inhibitor control. (A) Expression levels of miR-138 were measured by qRT-PCR; (B and E) the mRNA expression levels of RUNX3, T-bet and GATA3 were measured by qRT-PCR; (C) the protein expression levels of RUNX3 were measured by Western blotting. Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to ˇ-actin; (D) the protein level of IL-2, IFN-␥, IL-4 and IL-10 were measured by the ELISA method. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus inhibitor control.
3.4. Inhibition of miR-138 increases RUNX3 expression and regulate the cytokines related with Th1 and Th2 in CD4+ T cells from healthy controls The miR-138 inhibitor and inhibitor control were transfected into CD4+ T cells from healthy controls to investigate the effect of miR-138 inhibition in normal CD4+ T cells. After 48 h of transfection, the expression of miR-138 was detected using RT-PCR. The results showed that miR-138 expression was decreased significantly in CD4+ T cells transfected with the miR-138 inhibitor compared with the inhibitor control (Fig. 4A). The mRNA and protein levels of RUNX3 were detected using qRT-PCR and Western blotting. The results showed that the mRNA level of RUNX3 was significantly increased in CD4+ T cells transfected with the miR-138
inhibitor compared with the inhibitor control (Fig. 4B), which is consistent with the result of Western blotting (Fig. 4C). Next, the content of Th1 cell-related cytokines (IL-2, IFN␥) and Th2 cell-related cytokines (IL-4, IL-10) were measured using the ELISA method. Moreover, expression of the Th1related transcription factor T-bet and the Th2-related transcription factor GATA3 were measured by RT-PCR. The results showed that IL-2 and IFN-␥ were up-regulated and IL-4 and IL-10 were down-regulated significantly in CD4+ T cells transfected with the miR-138 inhibitor compared with the inhibitor control (Fig. 4D). Similarly, the expression of T-bet was increased and the expression of GATA3 was decreased in the inhibitor transfection of CD4+ T cells compared to the inhibitor control (Fig. 4E).
Fig. 5. Overexpression of miR-138 inhibits RUNX3 expression and regulates the cytokines related to Th1 and Th2 in CD4+ T cells from psoriasis patients. The CD4+ T cells from psoriasis patients were transfected with the miR-138 mimic or mimic control. (A) Expression levels of miR-138 were measured by qRT-PCR; (B and E) the mRNA expression levels of RUNX3, T-bet and GATA3 were measured by qRT-PCR; (C) the protein expression levels of RUNX3 were measured by Western blotting. Relative protein expression was quantified using Image-Pro Plus 6.0 software and normalized to ˇ-actin; (D) the protein levels of IL-2, IFN-␥, IL-4 and IL-10 were measured by ELISA. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus mimic control.
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Fig. 6. Overexpression of miR-138 regulates the deviation of Th1/Th2 in CD4+ T cells from psoriasis patients. The percentages of IFN-␥+ CD4+ T cells and IL-4+ CD4+ T cells in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control were measured by flow cytometry. (A and B) Flow cytometric analysis of Th1 in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control. The percentages of cells in Q2 region represent the percentage of CD4+ IFN-␥+ T cells. (C and D) Flow cytometric analysis of Th2 in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control. The percentage of cells in Q2 was the percentage of CD4+ IL-4+ T cells. (E) The frequency of Th1 and Th2 cells from psoriasis patients transfected with mimic or mimic control. (F) The frequency ratio of Th1 and Th2 in CD4+ T cells from psoriasis patients transfected with mimic or mimic control. Data are represented as the mean ± SD of three experiments. *P < 0.05 versus mimic control.
3.5. Overexpression of miR-138 inhibits RUNX3 expression and regulates the cytokines related to Th1 and Th2 in CD4+ T cells from psoriasis patients The miR-138 mimic and mimic control were transfected into CD4+ T cells from psoriasis patients to detect whether the overexpression of miR-138 could reverse the immune dysfunction. miR-138 expression was detected after 48 h of transfection using qRT-PCR. The results showed that miR-138 expression was significantly increased in CD4+ T cells transfected with miR-138 mimic compared with the mimic control (Fig. 5A). In addition, the mRNA and protein expression of RUNX3 was decreased significantly with miR-138 mimic transfection (Fig. 5B and C). Furthermore, the content of Th1 and Th2 cell-related cytokines was also detected, as well as the expression of Th1 related transcription factor T-bet and Th2-related transcription factor GATA3. The ELISA results showed that IL-2 and IFN-␥ were down-regulated and IL-4 and IL-10 were up-regulated significantly in CD4+ T cells transfected with miR-138 mimic compared with the mimic control (Fig. 5D). The qRT-PCR results showed that the expression of T-bet
was decreased and the expression of GATA3 was increased in the mimic-transfected CD4+ T cells when compared with the mimic control (Fig. 5E).
3.6. Overexpression of miR-138 regulates the balance of Th1/Th2 in CD4+ T cells from psoriasis patients The percentages of IFN-␥+ CD4+ T cells and IL-4+ CD4+ T cells in CD4+ T cells from psoriasis patients transfected with miR-138 mimic or mimic control were detected by flow cytometry. The results showed that the percentage of IFN-␥+ CD4+ T cells in the mimic group was decreased compared to those of the mimic control group (Fig. 6A and B). Moreover, the percentage of IL-4+ CD4+ T cells in the mimic group was increased compared to the mimic control group (Fig. 6C and D). Next, the ratio of Th1/Th2 in CD4+ T cells transfected with mimic or mimic control was calculated. The results showed that the ratio of Th1/Th2 in the mimic group was significantly lower than in the mimic control group (Fig. 6F).
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4. Discussion Psoriasis is one of the most common inflammatory and T cellmediated autoimmune diseases [1]. Tsoi et al. reported that RUNX3 is a susceptibility gene for psoriasis [15]. Consistently, we found that the expression of RUNX3 is significantly increased in CD4+ T cells from psoriasis patients in the present study. To investigate the role of RUNX3 in psoriasis, the noncoding region, 3 -UTR of RUNX3 was studied in this research. MiRNAs are noncoding RNAs that can suppress the expression of protein-coding genes by binding to the target sequence at the 3 -UTR of the target gene [17]. It has been reported that miRNAs play an important role in various diseases, including psoriasis [25]. After miRNA prediction and filtration using bioinformatic software programs, several miRNA were predicted to have target reaction with RUNX3. Among these miRNAs, we selected miR-138 to be the targeted miRNA fro RUNX3, which may be involved in the pathogenesis of psoriasis. In a previous study, miR-138 was observed to be downregulated in psoriasis [26]. It has been reported that the down-regulation of miR-138 is involved in several biological processes in different diseases. For example, miR-138 could induce cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma [27], could suppress ovarian cancer cell invasion and metastasis by targeting HIF-1a and SOX4 [28], and could inhibit glioblastoma cell proliferation in vitro and tumorigenicity in vivo via inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop [29]. Thus, we chose miR138 to be the targeted miRNA for RUNX3. In our study, the results showed miR-138 was decreased significantly in psoriasis patients compared with healthy controls, which was consistent with former study [26]. Furthermore, we detected a strong negative correlation between the expression level of miR-138 and the protein expression level of RUNX3 in CD4+ T cells from psoriasis patients. To verify the targeting reaction between miR-138 and RUNX3, the luciferase reporter vectors of wild type and mutant RUNX3 were constructed. The results showed that the overexpression of miR-138 inhibits luciferase expression when cells were transfected with wt-RUNX3 luciferase reporter system, but not in mut-RUNX3 groups. Moreover, the inhibition of miR-138 increases luciferase activity in wt-RUNX3 transfection group compared with mut-RUNX3 groups. These results demonstrate that RUNX3 is a target gene for miR-138. As has been previously reported, CD4+ T cells can differentiate down to Th1, Th2, Th17 and other type of T cells depending on cytokines, environmental signals and the nature of the encountered stimulus itself [30]. Ghoreschi et al. reported that the balance of Th1 and Th2 is associated with the development of psoriasis [31]. In addition, RUNX3 plays an important role in the differentiation of T cells and affects the balance of Th1/Th2 [16]. Thus, to investigate the role of miR-138 in T cell differentiation via targeting RUNX3, we detected the effect of miR-138 inhibition on expression of RUNX3 and deviation of Th1/Th2 in CD4+ T cells from healthy controls. The results showed that RUNX3 is significantly increased due to the inhibition of miR-138, which is similar to the physiological feature in psoriasis. Djuretic et al. reported that the expression of RUNX3 is related to transcription factor T-beta in the process of Th1 cells differentiation [32]. Moreover, RUNX3 augments Th1 cells and reduce Th2 cells by interacting with and attenuating GATA3 [33]. In the present study, we detected the expression level of Tbet and GATA3, as well as Th1-related cytokines (IL-2, IFN-␥) and Th2-related cytokines (IL-4, IL-10) in miR-138 inhibitor-transfected healthy CD4+ T cells. The results showed that the inhibition of miR-138 induces Th1-related cytokines and increased the expression of T-bet, whereas this inhibits Th2-related cytokines and also decreased the expression of GATA3. Taken together, these results suggest that the inhibition of miR-138 induce the deviation of Th1/Th2 by increasing RUNX3 expression, which may lead to the development of psoriasis.
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Recent studies have discovered several miRNAs that are potential therapeutic targets for psoriasis. Guinea-Viniefar et al. demonstrated that the inhibition of miR-21 ameliorated disease pathology in patient-derived psoriatic skin xenotransplants in mice and in a psoriasis-like mouse model [34]. Zhao et al. reported that the down-regulation of miR-210 improves immune dysfunction in CD4+ T cells of psoriasis patients [35]. In our study, we detected the effect of miR-138 overexpression in CD4+ T cells from psoriasis patients. The results showed that RUNX3 expression was inhibited due to the overexpression of miR-138. Moreover, the balance of Th1/Th2 was broken and shifted to overexpress of Th2-related cytokines. Furthermore, the result of flow cytometric analysis showed that the ratio of Th1/Th2 in CD4+ T cells decreased significantly after transfection with the miR-138 mimic. These results suggest that the overexpression of miR-138 regulates the balance of Th1/Th2, leading to immune dysfunction improvement via inhibition of the expression of RUNX3. In conclusion, our results demonstrated that the downregulation of miR-138 affects the expression of RUNX3, leading to an imbalance of Th1/Th2, which is involved in the pathogenesis of psoriasis. This study provides an important clue to help to elucidate the pathogenesis of psoriasis and implicates miR-138 as a potential therapeutic target for psoriasis.
Acknowledgments The study was supported by Science and Technology Research Key Project of The Education Department Henan Province (Grant NO. 13A320852), Science and Technology Research Plan in Xinxiang City (Grant No. ZG13022) and Health and Science Innovative Talents Project in Henan Province (Grant No. 201004159).
References [1] F.O. Nestle, D.H. Kaplan, J. Barker, Psoriasis N. Engl. J. Med. 361 (2009) 496–509. [2] M.A. Lowes, A.M. Bowcock, J.G. Krueger, Pathogenesis and therapy of psoriasis, Nature 445 (2007) 866–873. [3] C.E. Griffiths, J.N. Barker, Pathogenesis and clinical features of psoriasis, The Lancet 370 (2007) 263–271. [4] E. Lee, W.L. Trepicchio, J.L. Oestreicher, D. Pittman, F. Wang, F. Chamian, et al., Increased expression of interleukin 23 p19 and p40 in lesional skin of patients with psoriasis vulgaris, J. Exp. Med. 199 (2004) 125–130. [5] K. Ghoreschi, M. Rocken, Molecular and cellular basis for designing gene vaccines against inflammatory autoimmune disease, Trends Mol. Med. 9 (2003) 331–338. [6] K. Ghoreschi, P. Thomas, S. Breit, M. Dugas, R. Mailhammer, W. van Eden, et al., Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease, Nat. Med. 9 (2003) 40–46. [7] H. Tang, X. Jin, Y. Li, H. Jiang, X. Tang, X. Yang, et al., A large-scale screen for coding variants predisposing to psoriasis, Nat. Genet. 46 (2014) 45–50. [8] Garber K. Genetics, Deep exploration, Nature 492 (2012) S56–S57. [9] R.P. Nair, K.C. Duffin, C. Helms, J. Ding, P.E. Stuart, D. Goldgar, et al., Genomewide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways, Nat. Genet. 41 (2009) 199–204. [10] R. Yagi, I.S. Junttila, G. Wei, J.F. Urban Jr., K. Zhao, W.E. Paul, et al., The transcription factor GATA3 actively represses RUNX3 protein-regulated production of interferon-gamma, Immunity 32 (2010) 507–517. [11] K. Blyth, E.R. Cameron, J.C. Neil, The RUNX genes: gain or loss of function in cancer, Nat. Rev. Cancer 5 (2005) 376–387. [12] O. Brenner, D. Levanon, V. Negreanu, O. Golubkov, O. Fainaru, E. Woolf, et al., Loss of Runx3 function in leukocytes is associated with spontaneously developed colitis and gastric mucosal hyperplasia, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 16016–16021. [13] O. Fainaru, E. Woolf, J. Lotem, M. Yarmus, O. Brenner, D. Goldenberg, et al., Runx3 regulates mouse TGF-beta-mediated dendritic cell function and its absence results in airway inflammation, EMBO J. 23 (2004) 969–979. [14] Y. Naoe, R. Setoguchi, K. Akiyama, S. Muroi, M. Kuroda, F. Hatam, et al., Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer, J. Exp. Med. 204 (2007) 1749–1755. [15] L.C. Tsoi, S.L. Spain, J. Knight, E. Ellinghaus, P.E. Stuart, F. Capon, et al., Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity, Nat. Genet. 44 (2012) 1341–1348. [16] I.M. Djuretic, F. Cruz-Guilloty, A. Rao, Regulation of gene expression in peripheral T cells by Runx transcription factors, Adv. Immunol. 104 (2009) 1–23.
62
D. Fu et al. / Immunology Letters 166 (2015) 55–62
[17] V. Ambros, R.C. Lee, A. Lavanway, P.T. Williams, D. Jewell, MicroRNAs and other tiny endogenous RNAs in C. elegans, Curr. Biol. 13 (2003) 807–818. [18] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2009) 215–233. [19] H. Siomi, M.C. Siomi, Posttranscriptional regulation of microRNA biogenesis in animals, Mol Cell. 38 (3) (2010) 323–332. [20] E. Sonkoly, T. Wei, P.C. Janson, A. Saaf, L. Lundeberg, M. Tengvall-Linder, et al., MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoS One 2 (2007) e610. [21] N. Xu, P. Brodin, T. Wei, F. Meisgen, L. Eidsmo, N. Nagy, et al., MiR-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2, J. Invest. Dermatol. 131 (2011) 1521–1529. [22] G. Lerman, C. Avivi, C. Mardoukh, A. Barzilai, A. Tessone, B. Gradus, et al., MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R, PLoS One 6 (2011) e20916. [23] A. Ichihara, M. Jinnin, K. Yamane, A. Fujisawa, K. Sakai, S. Masuguchi, et al., microRNA-mediated keratinocyte hyperproliferation in psoriasis vulgaris, Br. J. Dermatol. 165 (2011) 1003–1010. [24] F. Meisgen, N. Xu, T. Wei, P.C. Janson, S. Obad, O. Broom, et al., MiR-21 is upregulated in psoriasis and suppresses T cell apoptosis, Exp. Dermatol. 21 (2012) 312–314. [25] T. Tomankova, M. Petrek, J. Gallo, E. Kriegova, MicroRNAs: emerging regulators of immune-mediated diseases, Scand. J. Immunol. 78 (2011) 129–141. [26] J.R. Zibert, M.B. Lovendorf, T. Litman, J. Olsen, B. Kaczkowski, L. Skov, MicroRNAs and potential target interactions in psoriasis, J. Dermatol. Sci. 58 (2010) 177–185.
[27] Y. Jin, D. Chen, R.J. Cabay, A. Wang, D.L. Crowe, X. Zhou, Role of microRNA-138 as a potential tumor suppressor in head and neck squamous cell carcinoma, Int. Rev. Cell Mol. Biol. 303 (2013) 357–385. [28] Y.M. Yeh, C.M. Chuang, K.C. Chao, L.H. Wang, MicroRNA-138 suppresses ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1alpha, Int. J. Cancer 133 (2013) 867–878. [29] S. Qiu, D. Huang, D. Yin, F. Li, X. Li, H.F. Kung, et al., Suppression of tumorigenicity by microRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme, Biochim. Biophys. Acta 1832 (2013) 1697–1707. [30] H. Kared, X. Camous, A. Larbi, T cells and their cytokines in persistent stimulation of the immune system, Curr. Opin. Immunol. 29 (2014) 79–85. [31] K. Ghoreschi, C. Weigert, M. Rocken, Immunopathogenesis and role of T cells in psoriasis, Clin. Dermatol. 25 (2007) 574–580. [32] I.M. Djuretic, D. Levanon, V. Negreanu, Y. Groner, A. Rao, K.M. Ansel, Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells, Nat. Immunol. 8 (2007) 145–153. [33] K. Kohu, H. Ohmori, W.F. Wong, D. Onda, T. Wakoh, S. Kon, et al., The Runx3 transcription factor augments Th1 and down-modulates Th2 phenotypes by interacting with and attenuating GATA3, J. Immunol. 183 (2009) 7817–7824. [34] J. Guinea-Viniegra, M. Jimenez, H.B. Schonthaler, R. Navarro, Y. Delgado, M.J. Concha-Garzon, et al., Targeting miR-21 to treat psoriasis, Sci. Transl. Med. 6 (2014) 225re221. [35] M. Zhao, L.T. Wang, G.P. Liang, P. Zhang, X.J. Deng, Q. Tang, et al., Up-regulation of microRNA-210 induces immune dysfunction via targeting FOXP3 in CD4(+) T cells of psoriasis vulgaris, Clin. Immunol. 150 (2014) 22–30.