Inflamm. Res. DOI 10.1007/s00011-015-0848-3

Inflammation Research

ORIGINAL RESEARCH PAPER

Characterization of microRNA expression profiling in peripheral blood lymphocytes in rats with experimental autoimmune uveitis Dadong Guo1 • Jiao Li1 • Zhengfeng Liu2 • Kai Tang2 • Huixin Song2 Hongsheng Bi1

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Received: 16 August 2014 / Revised: 18 June 2015 / Accepted: 24 June 2015  Springer Basel 2015

Abstract Objective and design We aimed to investigate the alterations of microRNA (miRNA) genomics in peripheral blood lymphocytes in experimental autoimmune uveitis (EAU) rats versus control samples. Materials/methods Six Lewis rats received interphotoreceptor retinoid-binding protein (IRBP) emulsion to induce EAU. On day 12, peripheral blood lymphocytes were isolated, and total RNAs were extracted. Using microarray analysis, we analyzed the aberrant miRNAs, validated the relevant expression of differentially expressed miRNAs, and predicted the possible miRNA targets and signaling pathways. Results The results indicated that 36 miRNAs were upregulated and 31 miRNAs were downregulated in EAU rats versus normal samples. Real-time quantitative PCR substantiated a high degree of confidence for the differentially expressed miRNAs, and miRNA analyses showed

Responsible Editor: Graham R. Wallace.

Electronic supplementary material The online version of this article (doi:10.1007/s00011-015-0848-3) contains supplementary material, which is available to authorized users. & Hongsheng Bi [email protected] 1

Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases, Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases in Universities of Shandong, Eye Institute of Shandong University of Traditional Chinese Medicine, Jinan 250002, China

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The Second Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan 250002, China

the differentially expressed miRNA targets were involved not only in the multicellular organismal process and developmental process, but also in T cell receptor signaling pathway, B cell receptor signaling pathway and so on. Conclusions Our findings show that the differentially expressed miRNAs in EAU rats were closely associated with immune signaling pathways and may be applied in early prevention, prognosis and possible therapy in uveitis, indicating that miRNAs play an important role in the development of uveitis. Keywords Experimental autoimmune uveitis  microRNA genomics  Rat  Expression profiling  Microarray

Introduction Uveitis is an autoimmune disease of the eye that refers to any of a number of intraocular inflammatory conditions, which could result in chronic inflammation of the eye and is an under-diagnosed and under-recognized medical condition that causes ocular pain and loss of vision. Uveitis is a kind of autoimmunity disease caused by complicated factors, which usually influences young people and accompanies more prolonged course [1]. To date, there is substantial evidence that T lymphocytes, which are key immune system cells, are involved in inflammatory processes and in the pathogenesis of autoimmune uveitis [2]. To investigate the detailed mechanism of the development and pathogenesis of uveitis, several animal models have been developed due to the difficulty in obtaining human eye inflamed tissues for experiments. Most of those models are induced by the injection of specific photoreceptors proteins (e.g., S-antigen, interphotoreceptor retinoid-

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binding protein, rhodopsin, recoverin, phosducin) [3]. In the above-mentioned models, the endotoxin-induced uveitis (EIU) is a well-characterized rat model for human acute uveitis [4]. After lipopolysaccharide (LPS) injection for about 4 h, resident cells (e.g., microglial cells, iris/ciliary body endothelial and epithelial cells) are activated and produce cytokines, chemokines and iNOS (inducible nitric oxide synthase) [5]. Further circulating inflammatory cells (e.g., polymorphonuclear leukocytes and macrophages) invade the anterior segment tissues of the eye [6, 7], and polymorphonuclear leukocytes or something like that promote the inflammation and cell injury of the ciliary bodies in this pathology [8]. Retinal S-antigen (SAg)- and interphotoreceptor retinolbinding protein (IRBP)-induced experimental autoimmune uveitis (EAU) are good models for studying the mechanisms involved in autoimmune diseases [9–12]. CD4 T cells that react to various self-antigens play a central role in the pathogenesis of uveitis, and the activation or inactivation of the CD4 T cells is controlled by costimulatory molecules. Currently, the animal model of EAU has been widely used for dissecting mechanisms and developing treatment strategies [13]. In EAU animal model, activated and sensitized Th1 and Th17 cells are usually considered to play an important role in initiation and maintenance of the intraocular inflammation [14, 15]. Cytokines play major role in maintaining lymphocyte homeostasis under conditions of health and disease. For uveitis, it is involved in increased levels of inflammatory cytokines and decreased levels of regulatory cytokines, including interleukin (IL)-17 [16, 17], IL-21 [18, 19], IL-10 [20, 21], IFN-c [22, 23] and so on. Meanwhile, it has also been reported that toll-like receptors (TLRs) are closely associated with the pathogenesis of uveitis, such as TLR2 [24, 25], TLR4 [24–28] and other TLRs [25]. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate messenger RNA (mRNA) at the posttranscriptional level. These highly conserved 22-mer RNAs regulate the translation of mRNA transcripts by binding principally to the 30 untranslated regions (30 UTR) of specific mRNAs to silence gene expression or promote degradation of target mRNAs [29–32]. miRNAs are complementary to the target mRNA sequence and only require complete complementarities of a 7-mer or 8-mer ‘‘seed sequence’’ for binding to occur. Currently, studies have shown that some diseases are closely associated with expression and regulation of miRNAs. miRNAs released by cancer cells within microvesicles can reach and bind to TLRs in surrounding immune cells and activate them in a paracrine loop. As a result, immune cells produce cytokines that increase cell proliferation and metastatic potential [33]; miRNAs could bind to TLRs to induce prometastatic inflammatory response [34] or influence the immune

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response to regulate the level of inflammatory cytokines [35]; IL-10-induced microRNA-187 could negatively regulate tumor necrosis factor (TNF)-a, IL-6, and IL-12p40 production in TLR4-stimulated monocytes [36]; mmu-ca65, a miRNA from mouse, could inhibit the release of proinflammatory cytokines, including TNF-a and IL-6, by regulating its target gene early growth response protein 1 in vivo [37]; miRNA let-7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis [38]. All these suggest the importance of miRNAs in regulating the development and pathogenesis of diseases. Recognition of miRNAs that are differentially expressed between uveitis rats and normal samples may help to identify those that are involved in uveitis progression and to establish the basis to uncover their pathogenic role. In the present study, the alteration of the miRNA profiling in peripheral blood lymphocytes in EAU rats was investigated, and the differentially expressed miRNAs were further validated by real-time quantitative PCR. Moreover, the differentially expressed miRNAs were also subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. In addition, the miRNAs and target genes predominating in the miRNA-gene regulatory networks were also investigated in an attempt to provide new insights into the understanding of the biological process of uveitis.

Materials and methods Induction of experimental autoimmune uveitis in rats Lewis rats (6–8 weeks) of specific pathogen-free grade were purchased from Beijing Vital River Laboratory Animal Co., Ltd. (Beijing, China) and were used for the induction of experimental autoimmune uveitis (EAU) model. All rats were fed and maintained as per the guidelines of Care and Use of Laboratory Animals published by China National Institute of Health. Interphotoreceptor retinoid-binding protein (IRBP) emulsion was prepared using 100 lg IRBP1177-1191 (residues 1177–1191, sequence ADGSSWEGVGVVPDV) peptide in Complete Freund’s Adjuvant (CFA, Sigma-Aldrich, St. Louis, MO.) supplemented with 50 ll of mycobacterium tuberculosis H37Ra (2.5 mg/ml; strain H37 Ra; Difco, Detroit, MI) in a total volume of 0.2 mL PBS solution. In the present study, five rats were separately immunized by an injection of 0.2 mL of IRBP emulsion in one footpad for the induction of EAU. Meanwhile, another five rats were treated as control by injecting equal volumes of saline solution with CFA supplemented with mycobacterium tuberculosis H37Ra.

Characterization of microRNA expression profiling in peripheral…

Pathological examination and graded criterion of EAU rats All rats were monitored using a slit-lamp biomicroscope (SL-D2, Topcon, Japan) every day. Severity of EAU in rats was scored on a scale of 0 (no disease) to 4 (maximum disease) according to the acknowledged criteria. 0 = normal; 1 = a few anterior chamber cells and slight iris-vessel dilatation; 2 = iris hyperemia, accompanied by limited pupil dilation, anterior chamber cells and a slight flare; 3 = a miotic, irregular, hyperemic and slightly damaged iris with a significant flare and cells; 4 = a seriously damaged and hyperemic iris, a miotic pupil and cloudy gellike aqueous humor [39, 40]. As to pathological alteration, eyes were removed on day 12 post-immunization, fixed in 4 % paraformaldehyde, and then embedded in paraffin. Sections (4 mm) were cut and stained with hematoxylineosin solution. The disease severity was determined for each eye and was scored on a scale of 0–4 in half-point increments according to a semi-quantitative system.

Measurement of CD4/CD8 ratio of peripheral blood lymphocytes To determine the alterations of CD4/CD8 ratio in peripheral blood lymphocytes for EAU rats versus control samples on day 12 after immunization, peripheral blood lymphocytes from both EAU rats and normal ones (control group) were, respectively, separated from whole blood with a Ficoll gradient method in accordance with the manufacturer’s instructions (Tianjin Haoyang Biological Manufacture Co. Ltd., China). After rinsing with PBS 3 times, both peripheral blood lymphocytes (1 9 106 cells in a 2 mL volume) were, respectively, added using FITCCD4? and PE-CD8? solutions, incubated at 37 C in a dark incubator for 30 min, followed by rinsing with PBS 2 times, and finally each sample was measured by a BD FACSVerse flow cytometer (USA).

RNA extraction, preparation of miRNAs and differentiation assay On day 12 after immunization with IRBP emulsification, peripheral blood lymphocytes from rats with EAU and normal rats (control group) were separated from whole blood using a Ficoll gradient according to the manufacturer’s instructions (Tianjin Haoyang Biological Manufacture Co. Ltd., China). Total RNA was harvested using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to the manufacturer’s instructions. After having passed RNA quantity measurement using a micro-

spectrophotometer (K5600, Beijing Kaiao Technology Development Co., Ltd.), the samples were labeled using the miRCURYTM Hy3TM/Hy5TM Power labeling kit and hybridized on the miRCURYTM LNA Array (v.18.0). Following the washing steps the slides were scanned using the Axon GenePix 4000B microarray scanner. Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. In all samples, the miRNAs that intensities C30 determined by microarray were chosen, and the replicated miRNAs were averaged for calculating normalization factor. Expressed data were normalized using the Median normalization and then significant differentially expressed miRNAs were identified through Volcano Plot filtering. Finally, hierarchical clustering was performed to show distinguishable miRNA expression profiling among samples.

Validation of miRNA expression by real-time quantitative PCR Real-time quantitative PCR was performed to verify the array results. In this study, rno-mir-20b-5p, rno-miR-17-5p, rno-miR-30b-5p, rno-miR-148b-3p, rno-miR-30b-3p, rnomiR-291a-5p, and RNU6B (internal control)-specific cDNAs syntheses were carried out using the Invitrogen Superscript ds-cDNA synthesis kit according to the manufacturer’s protocols. For PCR analysis, a quantitative PCR was performed using miScript SYBR-Green PCR Kit (Qiagen) (the primers were provided in Supplement Table 1). The reactions were incubated in a 384-well optical plate at 95 C for 10 min, followed by 40 cycles of 10 s at 95 C, 60 s at 60 C and then were held at 4 C. Expression analysis was performed in triplicate for each sample. Melting curve analysis (10 s at 95 C, 60 s at 60 C, and 15 s at 95 C) was used to confirm the specificity of the amplification reactions and the small nuclear RNA U6 was used as the normalization control. The miRNA expression level was quantified using ABI PRISM 7900 system (Applied Biosystems, Foster City, CA, USA). The relative expression of each miRNA was calculated using the 2-DDct method, with the ratio of the median expression sample among all rats with EAU/all normal (control) samples being used as the calibrator.

Target mRNA prediction of differential expression miRNAs To further understand the physiological functions and biological processes involved in these differentially expressed miRNAs, target gene prediction was performed based on miRNA/mRNA (messenger RNA) interactions to provide

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some molecular insight into the processes. Potential targets for miRNA action were predicted using putative targets generated from the relevant algorithms. Herein, the target mRNAs were predicted using rno-miR-291a-5p, rno-miR148b-3p, rno-miR-20b-5p, and rno-miR-30b-5p, and a combinatorial strategy was applied where target miRNAs were predicted for the differentially expressed genes using three algorithms, i.e., microcosm (http://www.ebi.ac.uk/ enright-srv/microcosm/htdocs/targets/v5/), miRanda (http:// www.microrna.org/) and miRDB (http://mirdb.org/miRDB/).

miRNA expression profiling arrays of EAU rats Total RNA of each sample was used for labeling and array hybridization. Double-strand cDNA (ds-cDNA) was synthesized from total RNA using an Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., USA). Briefly, ds-cDNA was incubated with 4 lg RNase A at 37 C for 10 min and cleaned using phenol: chloroform: isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a micro-spectrophotometer (K5600, Beijing Kaiao Technology Development Co., Ltd.). For Cy3 labeling of cDNA, the NimbleGen OneColor DNA labeling kit was used according to the manufacturer’s guideline detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). One microgram ds-cDNA was incubated for 10 min at 98 C with 1 OD of Cy3-9mer primer. Then, 100 pmol of deoxynucleoside triphosphates and 100 U of the Klenow fragment (New England Biolabs, USA) were added and the mix incubated at 37 C for 2 h. The reaction was stopped by adding 0.1 volume of 0.5 M EDTA, and the labeled dscDNA was purified by isopropanol/ethanol precipitation. Microarrays were hybridized at 42 C for 16–20 h with 4 lg of Cy3 labeled ds-cDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System-NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc., Madison, WI, USA). After being washed in an ozone-free environment, the slides were scanned using Agilent Scanner G2505C. Raw signal intensities were normalized using Robust Multichip Average (RMA) method by NimbleScan v2.5. For differentially expressed gene screening, statistical significance that passed Volcano Plot filtering (Fold change C2.0, p value B0.05) was chosen. Pathway analysis and GO Analyses were applied to determine the roles these differentially expressed genes played in these biological pathways or GO terms.

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Gene ontology (GO) analysis According to the bioinformatic techniques, target genes of aberrant miRNAs in rats with EAU compared with those in normal (control) rats were picked out. Moreover, these target genes were narrowed down according to the UniGene database which is specific to enterology. GO analysis was applied to organize genes into hierarchical categories and uncover the miRNA-gene regulatory network on the basis of biological process and molecular function [41]. Two-sided Fisher’s exact test and v2 test were used to classify the GO category, and the false discovery rate (FDR) was calculated to correct the p value. Herein, we chose GOs that had a p value of\0.05 and a FDR of \0.05. Within the significant category, the enrichment Re was given by Re = (nf/n)/(Nf/N), where nf is the number of flagged genes within the particular category, n is the total number of genes within the same category, Nf is the number of flagged genes in the entire microarray, and N is the total number of genes in the microarray.

KEGG pathway annotation based on miRNA expression profiling The predicted target genes were classified according to KEGG (Kyoto Encyclopedia of Genes and Genomes) function annotations to identify the pathways that were actively regulated by miRNAs. These aberrant miRNA targets were collected and were subjected to KEGG pathway annotation (http://www.genome.jp/kegg/). A twosided Fisher’s exact test and v2 test were used to classify the enrichment (Re) of pathway category, and the FDR was calculated to correct the p value. The enrichment Re was given by the same formula used in GO analysis, where nf and n represent the number of target genes and total genes, respectively, and Nf and N represent the number of genes among the entire differential miRNA corresponding target genes and the total number of genes on the pathway, respectively. We chose pathways that had a p value of \0.05 and an FDR of \0.05. The regulator pathway annotation was also performed on the basis of scoring and visualization of the pathways collected in the KEGG database (http://www.genome.jp/kegg/).

Statistical analysis All results were expressed as mean ± standard deviation (SD). Statistical analysis was performed with Student’s t test for comparison of two groups in microarray analysis

Characterization of microRNA expression profiling in peripheral…

in which differences with p \ 0.05 were considered statistically significant, and ANOVA for comparisons of expression of selected aberrant miRNAs in real-time quantitative PCR assay. In real-time quantitative PCR analysis, the differentially expressed miRNAs were designated as overexpressed if expression was [2.0-fold and as underexpressed if expression was \0.5-fold compared to normal samples while difference was considered significant at p \ 0.05, performed using SPSS software 16.0 (SPSS, Chicago, IL, USA).

Results Pathological examination of EAU rats Five rats were immunized with IRBP peptide emulsification and their eyes were examined using a slit-lamp biomicroscope every day until day 12 after immunization. Meanwhile, clinical scores for immunized rats were recorded every other day and the histopathological examination was performed on day 12 after immunization. As shown in Fig. 1, the peak inflammation was found on day 11–12 after immunization. Additionally, it was observed the opaque anterior chamber and obscured pupil, red reflex absent and hyphema in induced EAU rats on day 12 (Fig. 2b), whereas no apparent alterations in control ones (Fig. 2a). Meanwhile, the most severe intraocular inflammation was found on day 11–12 after immunization in EAU rats, as evidenced by inflammatory T lymphocytes infiltrating the vitreous and the ciliary body and damage to the retinal photoreceptor cell layer (Fig. 2f, g), whereas there were no apparent alterations in retina (Fig. 2h).

Alterations of CD4/CD8 ratio in EAU rats As shown in Fig. 3, it was found that on day 12, the levels of both CD4 and CD8 in total cell populations in EAU rats were changed (Fig. 3c) compared with those of control samples (Fig. 3b), and the ratio of CD4/CD8 was elevated in EAU rats versus control samples and was in a statistically difference response (Fig. 3d). Differential expression of miRNAs between EAU rats and normal samples Using the miRCURY LNA Array platform, we further evaluated the miRNA expression profiling in EAU and normal rats. The expression profiling of 278 miRNAs determined to be regulated between EAU and normal rats were sufficient to separate samples into biologically interpretable groups. Among these, there were 67 differentially expressed miRNAs in EAU rats versus control samples (Fig. 4), including 36 upregulated miRNAs and 31 downregulated miRNAs (Supplement Table 2). Validation of microarray data by real-time quantitative PCR analysis Six miRNAs among these filtered ones were validated to be significantly different between the EAU rats and normal samples (p \ 0.05 and p \ 0.01). As shown in Fig. 5, the levels of rno-miR-17-5p, rno-miR-148b-3p, rno-miR-20b5p and rno-miR-30b-5p were upregulated in EAU rats rather than normal ones (Fig. 5a), whereas the rno-miR291a-5p and rno-miR-30b-3p showed a downregulated expression pattern (Fig. 5b), which were in agreement with the results of microarray hybridization. Prediction of mRNAs for differential expression miRNAs

Fig. 1 The development of mean clinical score of 5 rats after immunization with IRBP peptide emulsification. Mean clinical score for rats was recorded every other day after immunization. Inset: the pathological score on day 11 after immunization for 5 rats; each diamond represents one eye from each EAU rat

To further understand the physiological functions and biological processes involved in these miRNAs during the development of EAU, target gene prediction was performed based on miRNA/mRNA interactions to provide some molecular insights into the processes. Using the relevant algorithms (i.e., microcosm, miRanda and miRDB), several genes were identified as potential targets of four miRNAs (Fig. 6). Twenty-eight mRNAs (including Atg12 and Becn1 associated with autophagy, IL21r associated with inflammation) were predicted as targets of rno-miR30b-5p, 35 mRNAs were predicted as targets of rno-mir20b-5p, in which Cdc37I1 and RGD1359108 were also predicated common targets of rno-miR-30b-5p and rnomir-20b-5p. Meanwhile, both rno-miR-30b-5p and rnomir-20b-5p were upregulated in EAU rats, whereas rno-

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D. Guo et al. Fig. 2 Typical photographs captured by a slit-lamp biomicroscope (a, b) on day 11 after immunization with IRBP peptide emulsification and histopatholgical features of the eyes of control rats (c–e) versus EAU rats (g–h). a rat in control group and b rat immunized with IRBP peptide emulsification. c, f iris; d, g ciliary body and e, h retina. Compared with normal architectures in control rats (c– e), inflammatory cells were observed in anterior chamber, iris (f) (arrows) and ciliary body (g) (arrows) in EAU rats, and mainly inflammatory T lymphocytes. Meanwhile, no apparent inflammatory T lymphocyte was found in retina in EAU rats (h). Magnification, 2009. Bar = 100 lm

mir-291a-5p was downregulated in EAU rats. Additionally, 17 genes were predicted as targets of rno-mir-291a-5p, and 14 genes were predicted as targets of rno-mir-148b-3p (Fig. 6). Microarray-based GO enrichment analysis GOs are international standardized classification system for gene annotations that provide insight into the molecular functions of genes in various biological processes [42]. The GOs cover three domains: biological process, cellular component and molecular function. On the basis of biological process, the genes were classified into 247 categories associated with upregulated miRNAs, including multicellular organismal process and developmental process (Fig. 7a). Meanwhile, 724 categories associated

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with downregulated miRNAs were identified, including cellular process and biological regulation (Fig. 7d). As regards the cellular component, genes associated with the overexpressed miRNAs were classified into 32 categories, including cytoplasmic part, extracellular region and mitochondrion (Fig. 7b), while genes associated with underexpressed miRNAs were classified into 47 categories, such as cell, cell part, intracellular and intracellular part (Fig. 7e). Based on the analysis of molecular function, it was noted that the genes could be classified into 13 categories associated with upregulated miRNAs, including protein kinase activity, iron ion binding and G-protein coupled receptor binding (Fig. 7c), whereas 106 categories associated with downregulated miRNAs were identified, including binding and protein binding (Fig. 7f).

Characterization of microRNA expression profiling in peripheral… Fig. 3 Representative flow cytometric data in EAU rats and control samples. a the gated lymphocytes in control samples after separation from peripheral blood cells; b CD4?CD8? cell population in EAU rats; c CD4?CD8? cell population in control rats; and d histogram analysis of the date presented as mean ± SEM of n = 3

KEGG pathway analysis based differentially expressed miRNAs targeted genes Based on the microarray results, the KEGG pathway was further applied to classify the putative miRNA targets. All predicted targets were clustered into 5 pathways for upregulated miRNA targeted genes (Fig. 8a) and 49 pathways for downregulated miRNA targeted genes (Fig. 8b), respectively. Among all these differentially regulated signaling pathways, we can note that downregulated miRNA targeted genes are mainly involved in the development of EAU, and the putative pathways include T cell receptor signaling pathway, chemokine signaling pathway, B cell receptor signaling pathway, Toll-like receptor signaling pathway, JAK-STAT signaling pathway, autophagy and so on. Discussion Uveitis is one of the most common eye diseases that can damage various people especially young ones. The development of uveitis accompanies by a series of pathological

alterations involved in a large-scale of gene upregulation and/or downregulation. However, limited understanding was made for the development and pathogenesis of uveitis in gene regulation until now. In our study, we found that after induction with IRBP emulsions in rats, uveitis occurred in rats accompanied by a typically inflammatory symptom in eyes. The results of histopathology reveal the infiltration of inflammatory cells in anterior chamber, iris and ciliary body in EAU rats. Considering uveitis is a systematic disease involving immune system, we further explored the alterations of CD4/CD8 ratios in peripheral blood lymphocytes and observed the elevated CD4/CD8 ratio. Sanz-Marco et al. [43] reported that CD4/CD8 ratio is also elevated in aqueous humor in patients with uveitis versus healthy subjects, and this result is in agreement with our findings, suggesting that the elevation of CD4/ CD8 ratio in uveitis samples is of great importance, and the alteration in CD4/CD8 ratio in peripheral blood lymphocytes is closely involved in the pathological process of uveitis.

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D. Guo et al. Fig. 4 Heat map and hierarchical clustering for differentially expressed miRNAs in rats with EAU and normal samples. Red indicates high relevant expression, and green indicates low relevant expression. Five normal rat samples and another five EAU rat samples were clustered in accordance with the expression profiling of 67 differentially expressed miRNAs between EAU rats versus normal ones, in which 36 miRNAs were upregulated and 31 miRNAs were downregulated (color figure online)

Increasing evidence confirmed that miRNAs can regulate gene expression at either post-transcriptional or translational level. Hundreds of miRNAs have been found in ocular tissues, and function as important regulators in physiological and pathological processes, including differentiation, development and regeneration after injury [44]. Thus, in the present study, we investigated the miRNA expression profiling during the development of rat

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uveitis. On being compared to that of normal rats, microarray analysis revealed a set of differentially expressed miRNAs in EAU rats with 38 significantly upregulated and 40 significantly downregulated miRNAs. Using real-time quantitative PCR technique, the tests of rno-mir-20b-5p, rno-miR-17-5p, rno-miR-30b-5p, rnomiR-148b-3p, rno-miR-30b-3p and rno-miR-291a-5p further validated the reliability of the results of microarray

Characterization of microRNA expression profiling in peripheral…

Fig. 5 Validation of microarray data using real-time quantitative PCR. Triplicate assays were done for each RNA sample and the relative amount of each miRNA was normalized to U6 snRNA. Statistically significant difference between EAU rats and normal samples is indicated by *p \ 0.05 or **p \ 0.01

hybridization. Based on these results, we speculate that these differentially expressed miRNAs play an important role in the development and pathogenesis of uveitis. Thus, the differentially expressed miRNAs are of importance in understanding the mechanism of development and

pathogenesis of unveitis. Nevertheless, to better understand the possible roles of these aberrant miRNAs, more experimental evidences should be carried out. Moreover, prediction of target mRNA for aberrant miRNAs further revealed the interactions between miRNAs and their mRNAs. miRNAs can predominantly bind to the 30 untranslated region of mRNAs to inhibit translation or to induce cleavage. Since the specific function of most miRNAs is unknown, it is necessary to find their relevant target mRNAs. Because of the difficulty of experimental identification of miRNA targets, several computational tools have been developed for predicting miRNA targets. In the present study, three algorithms, i.e., Microcosm, MiRanda and MiRdb, were used to predict the miRNA targets. We noted that upregulated rno-mir-30b-5p may regulate the gene expression of IL-21R, atg12, becn1 may regulate the gene expression associated with autophagy, and scn2n1, scn8a may regulate the gene expression related to voltage-gated sodium channel. Using Miranda and Mirdb, the possible target mRNAs of rno-mir-30b-5p may be correlated with IL-2 and toll-like receptor 4; using Miranda and Mirbase, the possible target mRNA of rnomir-30b-5p may be IL-10, and target mRNA correlated with downregulated rno-mir-291a-5p may be IL-17f, and these target mRNAs are involved in inflammation. In the meantime, the results are also consistent with those in GO

Fig. 6 A network of miRNAs rno-mir-30b-5p, rno-mir-20b5p, rno-mir-291a-5p and rnomir-148b-3p and their possible target genes. The red box nodes represent miRNA, blue cycle nodes represent mRNA. A combinatorial strategy was used where target miRNAs were predicted for the differentially expressed genes using microcosm, miRanda and miRDB

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D. Guo et al. Fig. 7 Gene ontology (GO) of the predicted targets for 65 differentially expressed miRNAs. Categorization of miRNA-target genes was performed in accordance with the biological process (a, d), cellular component (b, e) and molecular function (c, f). a– c indicated the categorization corresponding to upregulated miRNAs, and d–f indicated the categorization corresponding to downregulated miRNAs. The digit in the bracket was the amount of differentially expressed genes associated with the GO term

enrichment and KEGG pathway analysis. Importantly, our previous study has substantiated elevated levels of IL-10 and IL-17 in EAU rats compared with normal ones [21]. Ishida et al. [45] reported that experimental autoimmune uveoretinitis mice (B10.RIII) upregulated the expression of

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miRNA-142-5p, miRNA-21 and downregulated the expression of miRNA-182, accompanying the elevation of IL-17A and IL-17F in eyes, suggesting that aberrant miRNAs could regulate the development of uveitis. In our study, we observed that the expression of aberrant miRNAs

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was either increased (e.g., mir-148b-3p, mir-20b-5p, mir17-5p, mir-30b-5p) or decreased (e.g., mir-291a-5p, mir30b-3p), and we also found the aberrant expression of miRNAs accompanied by the abnormally expression of various proteins in serum in EAU rats [46]. Previous study has demonstrated that the mRNA levels of IL-17 and IFN-c were significantly upregulated in the retina in EAU rats. Similarly, the protein levels of both IL-17 and IFN-c were also markedly increased in serum in EAU rats [47], indicating that the same inflammatory trends occurred between ocular tissues and serum after EAU induction. Moreover, in a mouse model of endotoxin-induced uveitis, Xu et al. also reported that the expression of IFN-c in aqueous humor and serum was synchronously increased after induction of uveitis [48]. Thus, the dynamic alterations of the expression of aberrant proteins are closely correlated with the development of uveitis. Nevertheless, the microRNA expression profile varied widely between a rat model and a mouse model, and this difference may be attributed to the species variation and tissue difference. The GO project provides a controlled vocabulary to describe gene and gene product attributes in any organism. On the basis of predicted target genes of the aberrant miRNAs, GO analysis organized genes targeted by differentially expressed miRNAs into hierarchical categories based on biological process and then outlined the influences of miRNAs on EAU by means of abundant highenrichment GOs. It was noted that the initiation of EAU was stimulated by multicellular organismal process. The developmental process of EAU can be regulated by

upregulated miRNAs through protein kinase activity and G-protein coupled receptor and calmodulin signaling, and by downregulated miRNAs through the cellular process, biological regulation and regulation of biological process. Thus, the aberrantly expressed miRNAs may play an important role in the development and pathogenesis of uveitis through binding, protein binding and so on. All these may be involved in the participation of cytoplasmic part, extracellular region, mitochondrion, extracellular region part, cytosol and intracellular membrane-bounded organelle. Uveitis is a complicated and severe eye disease which involved in many molecules and signaling pathways. In our studies, the KEGG pathway analysis indicates that miRNA signaling upregulation pathways may be involved in porphyrin chlorophyll metabolism, glycine, serine, threonine metabolism and intestinal immune network for IgA production pathways. However, as regards miRNA signaling associated with downregulation pathways, it was found that most significantly regulated miRNAs have putative targets in many immune pathways, such as cell adhesion molecules, leukocyte transendothelial migration, T cell receptor signaling pathway, chemokin signaling pathway, B cell receptor signaling pathway, natural killer cell mediated cytotoxicity, toll-like receptor signaling pathway and so on. All these facts indicate that the interactions between downregulated miRNAs and mRNAs may play an important role in the pathologenesis of uveitis rather than upregulated miRNAs. Kaneko et al. [49] reported that no microRNA was significantly overexpressed in sympathetic

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D. Guo et al. Fig. 8 KEGG pathway analysis based on miRNA targeted genes. a The categorization of significant pathways targeted by upregulated miRNA; b The categorization of significant pathways targeted by downregulated miRNA

ophthalmia, a bilateral diffuse granulomatous uveitis of both eyes following trauma to one eye, whereas 27 miRNAs were markedly downregulated, suggesting that downregulated miRNAs may play a crucial role in the pathogenesis of sympathetic ophthalmia. Zhou et al. [50] also reported that only miR-155 expression was significantly decreased in peripheral blood mononuclear cells and dendritic cells from Behcet’s disease patients with active uveitis. Taken together, our findings substantiate that downregulated miRNAs may play a crucial role in the pathogenesis of uveitis either in experimental animals or in human being.

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Conclusion Our study has successfully distinguished the difference of miRNA expression profiling between rats with EAU and normal samples. The results indicate that 36 upregulated miRNAs and 31 downregulated miRNAs may be involved in the development and pathogenesis of uveitis in rats. The results of real-time quantitative PCR reveal that the differentially expressed miRNAs were in agreement with those determined by microarray hybridization. The related GO enrichment and KEGG pathway analyses also indicate that the differentially expressed miRNAs may be closely

Characterization of microRNA expression profiling in peripheral…

associated with the relevant immune pathway involved in T cell receptor signaling pathway, B cell receptor signaling pathway and toll-like receptor signaling pathway. These findings will facilitate the understanding that differentially expressed miRNAs may play a crucial role in pathogenesis of uveitis, and it may be applied in early prevention, prognosis and possible therapy in uveitis. Acknowledgements This study was supported by Doctoral Fund of Ministry of Education of China (20133731110004), the National Natural Science Foundation of China (81373826 and 81100658), and the Development Project of Science and Technology of Traditional Chinese Medicine of Shandong Province (2013ZDZK-083). Conflict of interest peting interests.

The authors declare that they have no com-

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