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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

Objective

MicroRNAs (miRNAs) function to fine-tune the control of immune cell signaling. It is well established that there are abnormalities in the interleukin-2 (IL-2)–related signaling pathways in systemic lupus erythematosus (SLE). The miR-31 microRNA has been found to be markedly underexpressed in patients with SLE, and thus the present study was undertaken to investigate the role of miR-31 in IL-2 defects in lupus T cells.

Methods

Expression levels of miR-31 were quantitated using TaqMan miRNA assays. Transfection and stimulation of cultured cells followed by TaqMan quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and reporter gene assays were conducted to determine the biologic function of miR-31. NF-AT nuclear translocation and expression were quantitatively measured using an ImageStream cytometer. Bioinformatics analysis, small interfering RNA (siRNA) knockdown, and Western blotting were performed to validate miR-31 targets and effects.

Results

The expression of miR-31 was significantly decreased in lupus T cells, and this was positively correlated with the expression of IL-2. Overexpression of miR-31 in T cells increased the production of IL-2 by altering NF-AT nuclear expression and IL2 promoter activity, while knockdown of endogenous miR-31 reduced IL-2 production. RhoA expression was directly repressed by miR-31 in T cells. Of note, siRNA-mediated knockdown of RhoA enhanced IL2 promoter activity and, consequently, up-regulated IL-2 production. RhoA expression was consistently up-regulated and negatively correlated with the levels of miR-31 in lupus T cells. Manipulation of miR-31 expression in lupus T cells restored the expression of IL-2 at both the messenger RNA and protein levels.

Conclusion

MicroRNA-31 is a novel enhancer of IL-2 production during T cell activation. Dysregulation of miR-31 and its target, RhoA, could be a novel molecular mechanism underlying the IL-2 deficiency in patients with SLE.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disorder characterized by chronic activation of the immune system and multiple immunologic phenotypes (1, 2). Many of the molecular aberrations identified in human lupus can explain certain established cell and cytokine defects, but the pathogenic mechanisms of SLE have not yet been fully elucidated.

It is well known that T cells in patients with SLE display a number of signaling abnormalities (3–6). Interleukin-2 (IL-2) is a multifunctional cytokine primarily produced by T cells and is essential for T cell activation, proliferation, and contraction (7). It has been reported that, in SLE T cells, the production of IL-2 is decreased and the transcriptional regulators responsible for the transcription or suppression of IL-2 are imbalanced (7, 8). The reduced production of IL-2 in SLE T cells was found to be caused by decreased levels of the transcription factors NF-κB and activator protein 1 (AP-1) and increased levels of the transcriptional repressor CREM, as well as abnormal expression of calcium/calmodulin-dependent protein kinase IV, protein phosphatase 2A, and other important mediators (9–12). Therefore, the molecular mechanisms that lead to decreased or absent expression of IL-2 in patients with SLE are manifold and the details are not yet fully understood.

MicroRNAs (miRNAs) are a class of 21–25-nucleotide single-stranded noncoding RNAs that act as key posttranscriptional regulators of gene expression (13, 14). In animals, miRNAs usually undergo imperfect basepairing with the 3′-untranslated region of their target genes, and can regulate target gene expression by translational inhibition or degradation of messenger RNA (mRNA) (15). The miRNAs play crucial roles in diverse biologic processes, including the regulation of immunologic functions and autoimmunity (16–19). Therefore, the dysregulation of miRNAs, and how this process may contribute to autoimmune disease, is an important area of research. Recent studies have provided growing evidence that the dysregulation of miRNAs contributes to the pathogenesis of SLE. For instance, it was shown that underexpression of miR-146a in lupus patients contributed to abnormal activation of the type I interferon pathway (20). In addition, miR-21, miR-148a, and miR-126 were reported to contribute to DNA hypomethylation in lupus CD4+ T cells (21, 22).

It has been reported that regulation of T cell activation and cytokine production can also occur by a miRNA-mediated pathway (23–25). Activation of the T cell–mediated immune response is associated with changes in the expression of specific miRNAs. The miR-146a microRNA, known to be induced upon T cell receptor (TCR) engagement in T lymphocytes, was found to impair IL-2 production and protect T cells from activation-induced cell death (26). In mice, miR-155 has been implicated as a positive regulator of cytokine production and T cell lineage choice (27, 28). However, to the best of our knowledge, whether the dysregulation of miRNAs contributes to the development of abnormalities in IL-2 production in lupus T cells is still unknown. We therefore hypothesized that miRNAs are involved in the molecular mechanisms leading to a defective IL-2 response in patients with SLE.

In our previous miRNA profiling study, we identified several miRNAs that were dysregulated in patients with SLE, and miR-31 was one of those most significantly down-regulated (20). In the present study, we first identified the underexpression of miR-31 in lupus T cells. We then determined that miR-31 acts as a positive regulator of IL-2 production in activated T cells by targeting RhoA. This candidate target gene increases the promoter activity of IL2 through the modulation of NF-AT nuclear expression; we found that the expression of RhoA was significantly higher in lupus T cells than in T cells of normal healthy volunteers. Thus, our results reveal that the underexpression of miR-31 contributes to the decreased production of IL-2 through the targeting of RhoA in lupus T cells. These findings provide additional insight into the role of miRNAs in the abnormalities of lupus, which might provide a rationale for the development of novel therapeutic strategies for SLE.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

Patients and normal healthy controls.

All samples from SLE patients were obtained from the Department of Rheumatology of Renji Hospital (Shanghai, China). All patients with SLE met at least 4 of the American College of Rheumatology 1982 revised criteria for SLE (29). The severity of disease was assessed with the SLE Disease Activity Index (30). The normal control group comprised healthy volunteers with no history of autoimmune disease or immunosuppressive therapy. Control subjects were matched with the patients for age and sex. All participants were from the Chinese Han population.

Peripheral blood samples (10 ml) were obtained from each subject. The samples were collected in tubes containing acid citrate dextrose Formula A. The study was approved by the Research Ethics Board of Renji Hospital, Shanghai JiaoTong University School of Medicine.

Isolation of CD3+ T cells.

Peripheral blood mononuclear cells (PBMCs) were separated from heparinized whole blood by density-gradient centrifugation, using Lymphoprep Ficoll-Paque Plus medium (GE Healthcare). CD3+ T cells were purified from fresh PBMCs by positive selection using magnetic CD3 microbeads (Miltenyi Biotec), in accordance with the manufacturer's protocol. The purity of the T lymphocytes was >95%, as analyzed on a FACSCalibur instrument (Becton Dickinson).

Cell culture and stimulation.

Purified T lymphocytes and Jurkat T cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, stimulated with phorbol myristate acetate (PMA) (50 ng/ml; Sigma-Aldrich) and ionomycin (1 μM; Sigma-Aldrich) for various lengths of time, at 37°C in a 5% CO2 atmosphere. Jurkat T cells, a T cell leukemia line, were grown in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin, at 37°C in 5% CO2. For ImageStream analysis, Jurkat T cells were stimulated with PMA (25 ng/ml) and ionomycin (1 μM).

MicroRNA mimics, small interfering RNAs (siRNAs), and antagomirs.

Small interfering RNAs and miRNA mimics were synthesized at Genepharma. The RhoA siRNA sequences were as follows: for RhoA siRNA-1, 5′-AAGATTATGACCGTCTGAGGC-3′; and for RhoA siRNA-2, 5′-AAGGATCTTCGGAATGATGAG-3′ (31). Antagomir-31 and its controls were obtained from Ribo Biology. A combination of gain-of-function (mimic-induced down-regulation) and loss-of-function (antagomir-induced up-regulation) experiments was used to demonstrate the miRNA–target relationships, which permitted functional analysis of miR-31.

Transfection.

Human primary T cells were rested in RPMI 1640 for 2 hours and then transfected with miRNA or siRNA oligonucleotides using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. Six hours after transfection, T cells were added to fresh complete medium (RPMI 1640 medium supplemented with 10% FBS). After 24 hours, the cells were stimulated with PMA and ionomycin for up to 24 hours at 37°C in an environment of 5% CO2.

For inhibition of miR-31, human primary T cells were resuspended in serum-free opti-MEM medium (Gibco) and transfected with 500 nM antagomir-31 or control scrambled antagomir. After 6 hours of transfection, RPMI medium supplemented with 500 nM antagomir was added, and 24 hours later, the cells were stimulated in the same manner as described above. For ImageStream analysis, Jurkat T cells were transfected with miR-31 mimic (200 nM) or control mimic (200 nM), using the Neon Transfection System (Invitrogen) according to the manufacturer's protocol.

Antibodies and staining.

The NF-AT protein was visualized by indirect labeling using a Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences). Briefly, cells were collected and washed with 1× cold phosphate buffered saline after drug treatment. Cells were then fixed and permeabilized for 20 minutes at 4°C in fixation/permeabilization solution containing 4% paraformaldehyde. Anti–NF-ATc1 (BioLegend) was diluted 1:100 in buffer (1× BD Per/Wash buffer; BD Biosciences) containing FBS and saponin, and was then added to wells containing 1 × 106 cells. Samples were incubated for 30 minutes at 4°C. Primary antibody was removed, and a 1:100 dilution of secondary, fluorescein isothiocyanate–conjugated F(ab′)2 fragment donkey anti-mouse IgG antibody (Jackson ImmunoResearch) was added, followed by incubation for 30 minutes at 4°C in the dark. Cells were washed with 1× BD Per/Wash buffer and resuspended in staining buffer containing 0.1% bovine serum albumin. Nuclear red DRAQ5 (eBioscience) was added to all samples for nuclear staining prior to ImageStream analysis.

ImageStream cytometry analysis.

Cells fluorescently probed with the anti–NF-AT antibody and DRAQ5 were subjected to ImageStream analysis. In total, up to 20,000 events were collected for each sample on an ImageStream cytometer, using a laser excitation wavelength of 488 nm. The collected images of the fixed cells were analyzed with ImageStream Data Exploration and Analysis software. NF-AT nuclear translocation was quantitatively measured using similarity analysis on in-focus single cells, and expression levels were analyzed as the median fluorescence intensity.

Quantitative polymerase chain reaction (PCR).

Total RNA was isolated with TRIzol reagent (Invitrogen). To quantify miRNAs, samples of RNA (20 ng) were reverse transcribed using a TaqMan miRNA reverse transcription (RT) kit (Applied Biosystems). TaqMan miRNA assays (Applied Biosystems) were used for real-time PCR, in accordance with the manufacturer's recommendations, and RNU48 was used as an endogenous control to normalize the expression values. For quantitative RT-PCR, 500 ng of total RNA was reverse transcribed using a PrimeScript RT reagent kit (Takara). Complementary DNA (cDNA) was amplified by real-time PCR with SYBR Green (SYBR Premix Ex Taq RT-PCR kit; Takara); the ribosomal protein L13A (RPL13A) gene was used as an internal control to normalize the amounts of cDNA. The TaqMan and SYBR Green assays were performed in duplicate on a 7900HT Fast Real-Time PCR instrument (Applied Biosystems). The relative expression levels were calculated using the 2math image threshold cycle method.

The following custom primers were used for SYBR Green–based real-time PCR: for RPL13A, forward 5′-CCTGGAGGAGAAGAGGAAAGAGA-3′ and reverse 5′-TTGAGGACCTCTGTGTATTTGTCAA-3′; for RhoA, forward 5′-TCTTCGGAATGATGAGCAC-3′ and reverse 5′-CTTTGGTCTTTGCTGAACAC-3′; and for IL2, forward 5′-TGCCACAATGTACAGGATGC-3′ and reverse 5′-GCCTTCTTGGGCATGTAAAA-3′.

Cloning of reporter constructs, transient transfection, and luciferase assays.

The pGL3-Basic Luciferase Reporter vector (Promega) was used to generate IL2 promoter reporter constructs. Briefly, the 884-bp promoter region (+59 to −825 bp) was amplified by PCR from genomic DNA. The primers used were as follows: forward 5′-CCGACGCGTCCATTCATAGTGTCCCAGGTG-3′ and reverse 5′-CCGCTCGAGCATTGTGGCAGGAGTTGAG-3′. The forward and reverse primers created Mlu I sites and Xho I sites, respectively, and the PCR products were ligated into these sites in the pGL3-Basic plasmid, in accordance with the manufacturer's instructions. Jurkat T cells were seeded at 1 × 106 cells/well in a 24-well plate and transfected 2 hours later. The pGL3-Basic luciferase reporter plasmids (1 μg) containing the IL2 promoter (as described above) and the miRNA oligonucleotides or siRNAs, together with 50 ng of a pRL-Basic-Luc vector used to normalize transfection efficiency, were cotransfected into Jurkat T cells using Lipofectamine 2000 (Invitrogen). After a 24-hour recovery period, transfected cells were either left untreated or stimulated for 24 hours with PMA and ionomycin. Luciferase activity was then assessed using a Dual Luciferase reporter assay system (Promega), carried out using a CENTRO XS3 LB 960 instrument (Berthold Technologies) in accordance with the manufacturer's protocol. The ratio of Renilla luciferase to firefly luciferase was obtained for each well. All experiments were performed in triplicate.

Enzyme-linked immunosorbent assay (ELISA).

The amount of IL-2 protein secreted into the cell culture supernatant was quantified using commercially available ELISA kits (Xi Tang Biology). ELISAs were carried out in accordance with the manufacturer's protocol.

Western blotting.

T cells were seeded at 5 × 106 cells/well in a 6-well plate and transfected with miRNA oligonucleotides to a final concentration of 100 nM per well. Six hours after transfection, the T cells were added to fresh complete medium. Two days after transfection, the cells were lysed and proteins were extracted. The supernatants were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with the appropriate antibodies, followed by protein detection with Luminol/Enhancer Solution (Pierce). Western blotting for RhoA and α-tubulin was performed using a mouse anti-RhoA antibody (1:200; Santa Cruz Biotechnology) and rabbit anti–α-tubulin antibody (1:5,000; Santa Cruz Biotechnology), respectively. The volume tools of Quantity One software (Bio-Rad) were used to quantitate the protein bands, according to the manufacturer's instructions.

Statistical analysis.

Data were analyzed using Prism software (version 4.03; GraphPad). The nonparametric Mann-Whitney test was used to compare gene expression between 2 groups, while the Student's unpaired t-test was used to compare reporter gene activity and fluorescence intensity. P values (2-tailed) less than 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

Correlation of decreased miR-31 expression with low IL-2 expression in lupus T cells.

In our previous experiments, we profiled the expression of 156 miRNAs in peripheral blood lymphocytes and identified 42 miRNAs that were expressed differentially between patients with SLE and normal controls (20). Among these miRNAs, miR-31 was significantly down-regulated in SLE patients compared to normal controls. Because the expression patterns of miRNAs can provide hints about their possible functions, we measured the expression level of miR-31 in T cells, B cells, and monocytes isolated from the PBMCs of 3 normal healthy volunteers. As shown in Figure 1A, miR-31 was selectively expressed in T cells, which indicates that miR-31 might play a role in T cell function.

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Figure 1. Decreased expression of microRNA-31 (miR-31) in patients with systemic lupus erythematosus (SLE), and positive correlation with interleukin-2 (IL-2) expression in lupus T cells. A, Expression of miR-31 in T cells, B cells, and monocytes from normal healthy donors was determined by TaqMan quantitative polymerase chain reaction (PCR). Results are the mean ± SEM relative expression (n = 3), normalized to the values in B cells. B, Expression of miR-31 in T cells was independently verified by TaqMan quantitative PCR in peripheral blood samples from 32 patients with SLE and 11 normal controls (NC). Results are the mean ± SEM relative expression. C, Primary T cells from normal healthy donors were stimulated with phorbol myristate acetate and ionomycin, and miR-31 levels were measured by TaqMan quantitative PCR at different time points after stimulation. Bars show the mean ± SEM from 3 independent experiments, with results normalized to the levels of RNU48. D, Linear correlation analysis was performed to assess the correlation between IL-2 and miR-31 relative expression in activated T cells of patients with SLE (n = 15). P value was determined by Mann-Whitney U test.

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We then further investigated the expression of miR-31 in T cells obtained from 32 patients with SLE and 11 normal control subjects. We found that miR-31 was significantly down-regulated in the SLE patients compared with normal controls (P < 0.0001) (Figure 1B) (clinical data are available from the corresponding author upon request).

T cells from lupus patients display numerous signaling abnormalities that may contribute to the pathogenesis of SLE (5, 6). We therefore wanted to determine the function of miR-31 in the skewed expression of T cell–related cytokines in SLE. We first determined whether TCR engagement affected miR-31 expression. CD3+ T lymphocytes freshly purified from normal healthy donors were stimulated with PMA and ionomycin. In a time-course study of these cells, as shown in Figure 1C, we found that miR-31 expression was induced after stimulation of the cells, reaching a peak level at ∼12 hours.

Given the fact that T cell activation through the TCR results in enhanced induction of IL-2 and there is a well-established IL-2 defect in lupus T cells, we further investigated the expression of miR-31 and IL2 in activated lupus T cells (n = 15). Our results showed a positive correlation between miR-31 expression and IL2 mRNA levels (Figure 1D).

Role of miR-31 as a positive regulator of IL-2 expression.

The positive correlation between the expression of miR-31 and the expression of IL-2 in lupus T cells prompted us to investigate whether miR-31 could modulate IL-2 production. To address this question, human primary T cells were transfected with miR-31 mimic or control mimic and activated with PMA and ionomycin for 24 hours. IL2 mRNA levels were then measured by RT-PCR, with results normalized to the values for the housekeeping gene RPL13A. IL-2 protein levels in the supernatants were also measured by ELISA.

As shown in Figure 2A, transfection of T cells with the miR-31 mimic increased the expression of IL-2 at both the mRNA and protein levels, while silencing of the endogenous miR-31 via transfection with the inhibitory oligonucleotide antagomir-31 decreased IL-2 production. The successful ectopic expression of miR-31 and the silencing of miR-31 in primary T cells was verified by TaqMan quantitative PCR (details available from the corresponding author upon request), which showed that overexpression of miR-31 resulted in a 50-fold increase in miR-31 expression, while inhibition of miR-31 led to a 25-fold decrease in miR-31 expression.

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Figure 2. Regulation of interleukin-2 (IL-2) production by microRNA-31 (miR-31) via its effects on nuclear NF-AT expression and IL2 promoter activity. A, The fold change in IL2 mRNA expression (left) and the IL-2 levels detected in culture supernatants by enzyme-linked immunosorbent assay (right) were determined in activated primary T cells from normal healthy donors after incubation with miR-31 mimic or control (Ctrl) mimic or with antagomir-31 or scrambled control. Bars show the mean ± SEM results in 4 representative donors per group. B, Jurkat T cells were cotransfected with miR-31 mimic or control mimic plus the IL2 promoter-luc reporter plasmid; pGL3-Basic-Luc was used for normalization of transfection. Cells were left unstimulated (0 hours) or stimulated with phorbol myristate acetate and ionomycin for 24 hours, and the relative IL2 promoter activity was measured by dual luciferase assay. Bars show the mean ± SEM of 3 independent experiments. C, Histograms show the relative expression of NF-AT in Jurkat T cells transfected with miR-31 mimic or control mimic. The staining intensity of NF-AT was determined in control-transfected cells before stimulation and in miR-31– and control-transfected cells at 48 hours after stimulation (top). Representative images of Jurkat T cells display the effect of miR-31 on nuclear NF-AT expression at 48 hours after stimulation, compared to controls, as determined by nuclear red DRAQ5 and fluorescein isothiocyanate (FITC) staining (bottom). D, The fold change in median fluorescence intensity of NF-AT staining was determined in activated Jurkat T cells overexpressing miR-31 mimic or control mimic. Bars show the mean ± SEM fold change in miR-31–transfected or control-transfected cells after 48 hours of stimulation relative to that in unstimulated controls (n = 3 per group). ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus unstimulated control.

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We then performed luciferase reporter assays to address whether miR-31 regulates IL2 promoter activity. The IL2 promoter was cloned into a luciferase reporter vector (IL-2-luc). Jurkat T cells were cotransfected with IL-2-luc, along with miR-31 or a control, and were stimulated with PMA and ionomycin. As shown in Figure 2B, miR-31 had little or no effect on IL2 promoter activity in unstimulated T cells, but dramatically increased IL2 promoter activity in the T cells after 24 hours of stimulation. Overexpression of miR-31 resulted in a dose-dependent elevation of IL2 promoter luciferase activity (details available from the corresponding author upon request). Taken together, these data suggest that miR-31 promotes IL-2 production through enhancement of IL2 promoter activity in activated T cells. This regulatory relationship might be important in patients with SLE.

Promotion of nuclear NF-AT expression by miR-31.

The IL2 promoter region possesses multiple binding sites for the transcription factor NF-AT, whose activation in response to extracellular stimulation has been shown to be required for IL-2 production in Jurkat T cells (32). We thus wondered whether miR-31 exerts its effect on IL-2 production through the regulation of NF-AT expression. To address this question, we used ImageStream cytometry to examine NF-AT nuclear translocation and expression upon induction of miR-31 expression in activated Jurkat T cells.

As shown in Figures 2C and D, the majority of the NF-AT translocated to the nucleus of Jurkat T cells after stimulation with PMA (25 ng/ml) and ionomycin (1 μM) for 48 hours. Overexpression of the miR-31 mimic significantly increased nuclear NF-AT expression in activated Jurkat T cells, when compared to cells transfected with the control mimic. The effect of miR-31 on nuclear NF-AT activity was further supported by chromatin immunoprecipitation assay (details available from the corresponding author upon request), which showed that overexpression of miR-31 in T cells increased the amount of immunoprecipitated IL2 promoter segment.

Targeting of RhoA by miR-31 in T cells.

To gain insight into the molecular mechanisms of IL-2 regulation by miR-31, we used bioinformatic tools to identify the potential targets of miR-31. Utilizing the miRGen database (available at http://www.diana.pcbi.upenn.edu/miRGen/v3/miRGen.html), which integrates TargetScan, PicTar, and MiRanda analyses, we generated a list of predicted miR-31 targets. Focusing on genes relevant to T cell activation and IL-2 production, we identified RhoA as a candidate target of miR-31, because it has been reported to modulate IL2 gene expression in T cell activation (33). In addition, RhoA has been demonstrated to be a target gene of miR-31 in a study using a reporter system in MDA-MB-231 cells (34). To evaluate whether miR-31 inhibits the expression of RhoA in T cells, we transfected primary T cells with miR-31 mimic or control mimic, and then measured the expression of RhoA by both RT-PCR and Western blotting. Results of RT-PCR demonstrated that miR-31 inhibited the levels of RhoA mRNA (Figure 3A). Furthermore, Western blotting revealed that miR-31 transfection of T cells resulted in a reduction in RhoA protein (Figure 3B).

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Figure 3. Identification of microRNA-31 (miR-31) as a negative regulator of RhoA expression. A, RhoA mRNA expression levels in activated T lymphocytes transfected with either miR-31 mimic or control (Ctrl) mimic were analyzed by reverse transcription–polymerase chain reaction (RT-PCR). Bars show the mean ± SEM of 3 independent experiments. B, RhoA protein expression was analyzed by immunoblotting of primary T cells at 48 hours after transfection of the cells with miR-31 mimic or control mimic (n = 3). Values below blots show the ratio of RhoA to α-tubulin. C, The expression of RhoA in T cells from patients with systemic lupus erythematosus (SLE) (n = 32) and normal controls (NC) (n = 11) was measured by RT-PCR. Bars show the mean ± SEM. D, Linear correlation analysis was performed to assess the correlation between the expression of RhoA and the expression of miR-31 in primary T cells of patients with SLE (n = 32). P value was determined by Mann-Whitney U test.

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Given that miR-31 expression levels in SLE patients were significantly lower than those in normal healthy controls, we investigated whether RhoA expression was increased in SLE T cells. We examined T cells obtained from 32 patients with SLE and 11 normal healthy controls, and found that RhoA mRNA was significantly up-regulated in SLE T cells (Figure 3C). Furthermore, a linear correlation analysis demonstrated that the expression of RhoA mRNA correlated negatively with the expression of miR-31 in primary T cells of patients with SLE (Figure 3D). Taken together, these data suggest that miR-31 targets RhoA in human primary T cells, and that RhoA mRNA expression is significantly higher in lupus T cells than in normal healthy T cells.

Knockdown of RhoA expression leading to enhanced IL2 promoter activity and IL-2 production.

Considering the observed up-regulated expression of RhoA mRNA and down-regulated expression of miR-31 and IL-2 in lupus T cells, along with the regulatory role of RhoA in modulating IL2 gene expression in T cells (33), we hypothesized that miR-31 regulates the expression of IL-2 in T cells by targeting RhoA. If this is the likely scenario, then the inhibition of RhoA expression would have an effect equivalent to that caused by the overexpression of miR-31. To test this, we used RNA interference to knock down the expression of RhoA. Transfection of cells with RhoA siRNA for 48 hours significantly reduced the expression of RhoA at both the mRNA and protein levels (details available from the corresponding author upon request), an effect similar to that of overexpression of the miR-31 mimic.

We then transfected primary T cells with siRNA or miR-31 mimic for 48 hours, followed by stimulation with PMA and ionomycin for 24 hours, and measured the expression levels of IL-2. Consistent with the observed effects of the miR-31 mimic, knockdown of RhoA led to a marked increase in the expression of both IL2 mRNA and IL-2 protein (Figures 4A and B). We then evaluated the effect of RhoA siRNA on the activity of the IL2 promoter, and found that knockdown of RhoA enhanced the activity of the IL2 promoter (Figure 4C).

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Figure 4. Effect of small interfering RNA (siRNA)–mediated knockdown of RhoA in regulating interleukin-2 (IL-2) production and IL2 promoter activity. A and B, The relative expression of IL2 determined by quantitative polymerase chain reaction (A) and the IL-2 levels in culture supernatants determined by enzyme-linked immunosorbent assay (B) were assessed in activated primary T cells at 24 hours after transfection of microRNA-31 (miR-31) mimic or control (Ctrl) mimic or after transfection with siRNAs 1 and 2 (siR-1 and -2). Bars show the mean ± SEM results in samples from 3 representative normal healthy donors. C, Jurkat T cells were cotransfected with reporter vectors containing the IL2 promoter and RhoA siRNA, miR-31 mimic, or control mimic, together with pGL3-Basic-Luc for normalization of transfection, and the luciferase activity of miR-31– and siRNA-transfected cells, relative to controls, was assessed by dual luciferase assay. Bars show the mean ± SEM of 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.01, versus control.

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Correction of IL-2 production in SLE T cells by ectopic miR-31 expression.

Given that miR-31 was induced in activated T cells and that miR-31 regulated IL-2 production by targeting RhoA, we investigated the expression levels of miR-31, RhoA, and IL-2 in activated T cells isolated from 15 patients with SLE and 10 normal healthy controls (clinical data available from the corresponding author upon request). The expression of miR-31 and IL-2 in activated T cells from patients with SLE was lower than that observed in activated T cells from normal controls, while the expression of RhoA was higher in patients with SLE compared to controls (Figures 5A–C).

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Figure 5. Role of microRNA-31 (miR-31), RhoA, and interleukin-2 (IL-2) in activated T cells from patients with systemic lupus erythematosus (SLE). A and B, The relative expression of miR-31 determined by TaqMan quantitative polymerase chain reaction (PCR) (A) and the relative expression of RhoA determined by reverse transcription–PCR (B) was assessed in activated T cells from 15 patients with SLE and 10 normal controls (NC). C, IL-2 protein levels were determined by enzyme-linked immunosorbent assay (ELISA) in supernatants of activated T cells from 12 patients with SLE and 10 normal controls. D, IL-2 expression in culture supernatants of activated T cells from patients with SLE (n = 3) was determined by ELISA after transfection of the cells with miR-31 mimic or control (Ctrl) mimic. Bars show the mean ± SEM. P values were determined by Mann-Whitney U test.

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Furthermore, we explored whether the manipulation of miR-31 levels could affect IL-2 production in T cells from patients with SLE. We transfected lupus T cells with miR-31 mimic or control mimic. After 24-hour stimulation with PMA and ionomycin, we observed enhanced IL-2 protein expression in the activated lupus T cells transfected with miR-31 mimic (Figure 5D). This indicates that manipulation of miR-31 levels in the T cells of patients with SLE could potentially rescue the defects in IL-2 production.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

SLE is a complex autoimmune disease that has diverse cellular and molecular aberrations. It has been shown that T cells in patients with SLE, as well as in murine models of SLE, exhibit defects in IL-2 synthesis, which contributes to the increased susceptibility to infections, decrease in activation-induced cell death, and subsequent extended survival of autoreactive lymphocytes (35). Furthermore, recent studies have revealed a vital role of IL-2 in the homeostatic control of regulatory T cells (36, 37). The expression of IL-2 is tightly regulated in multiple ways, such as epigenetically and by multiple transcription factors that bind to distinct sites on the IL2 promoter (38, 39). Our study confirmed that miR-31 might be a novel mechanism of fine regulation of IL-2 production.

We first established that miR-31 was significantly down-regulated in lupus T cells and that its target gene, RhoA, was overexpressed, which contributed to the IL-2 deficiency in lupus. These novel findings extend the role of miRNAs as a complement of transcription; we found that miR-31 acts as a regulatory factor involved in the abnormal signaling pathways of the IL-2 defect in lupus T cells.

Transcriptional dysregulation of IL2 has been demonstrated in T cells from patients with SLE. Deficiencies in the TCR and in the expression of NF-κB and AP-1, and, especially, the abnormal expression of CREM and CREB, have been implicated in this phenomenon (7, 40, 41). We showed that the expression levels of miR-31 positively correlated with the levels of IL-2 in lupus T cells. Down-regulated miR-31 expression resulted in high levels of RhoA, a target of miR-31 in SLE. RhoA, a small GTPase that is well characterized for its role in cytoskeletal rearrangement, has been shown to inhibit IL-2 production in T cells and regulate the IL2 promoter in an NF-AT–dependent manner (33, 38). Our investigation showed that siRNA-mediated knockdown of RhoA enhanced the IL2 promoter activity and IL-2 production in T cells. Inhibition of RhoA expression had an effect similar to that of overexpression of miR-31. We conclude that miR-31 regulates IL-2 production in T cells by targeting RhoA.

The underlying mechanism of IL-2 dysregulation in SLE T cells is highly complicated and may involve participation of multiple layers of regulation. In our study, we have, for the first time, identified an excessive level of RhoA in lupus T cells, which arises because of a deficiency in miR-31. The induction of RhoA may cause impaired IL2 promoter activity, which further leads to defective IL-2 production in SLE T cells. This finding is consistent with the findings from a previous study, in which it was demonstrated that RhoA is capable of suppressing the ability of NF-AT to activate the transcription of IL2 (33).

However, earlier observations indicated that other epigenetic mechanisms, such as histone acetylation, may also influence IL2 promoter activity (33, 38). Bodor et al showed that interaction of NF-AT with the inducible cAMP early repressor prevented the recruitment of the CREB binding protein/p300 histone acetylase complex to the promoter and the inhibition of transcription (42–44). Splenocytes from autoimmune MRL-lpr/lpr mice were characterized as hypoacetylated for the H3 and H4 histones (45). Another investigation demonstrated global histone H3/H4 hypoacetylation in active lupus CD4+ T cells as compared with control cells. Furthermore, the degree of histone H3 acetylation was negatively correlated with increased disease activity in lupus patients (46). We hypothesize that deficiency of miR-31 may also partially contribute to the histone H3/H4 hypoacetylation in lupus T cells via the targeting of RhoA. A potential association of RhoA with the regulation of histone acetylation is thus worth further exploration.

The human miRNA miR-31 is relatively well characterized and is located at chromosome 9p21.3 (47). We demonstrated that miR-31 was down-regulated in patients with SLE and could not be fully induced in the lupus T cell activation process, as compared with that in normal healthy control subjects. However, it is not clear what factors cause the dysregulation of miR-31 expression in SLE. Further research is required to investigate the possible mechanisms of the defects in miR-31 expression in lupus. Additional in silico predictions and animal models will help advance this work.

Gene therapy approaches involving the delivery of genes that encode immunomodulatory molecules, such as cytokines, have been used extensively in animal models of SLE (48–51). There is, however, emerging evidence to support the notion that miRNAs hold great promise as a novel therapeutic target and clinical biomarker for human diseases. The miRNAs are capable of regulating a network of genes in a mild, but long-lasting, manner, potentially circumventing the effect of by-product toxicity that is normally observed in current lupus therapies. In addition, the application of miRNA-based disease intervention and prevention strategies has the potential to considerably reduce the economic burden on patients with SLE. Given the importance of IL-2 in the immune response and the consequences of its deficiency in SLE T cells, we propose that an approach involving replenishment of miR-31 would restore normal IL-2 production. Interestingly, when the miR-31 mimic was introduced into lupus T cells, the expression of IL-2 was increased. These results suggest that miR-31 levels could be manipulated to provide useful therapeutic interventions for SLE.

In conclusion, we have shown that underexpression of miR-31 contributes to the abnormal production of IL-2 in lupus T cells via its targeting of RhoA, which results in altered nuclear NF-AT expression and IL2 promoter activity. Our findings suggest that miR-31 could serve as a potential therapeutic target for the treatment of SLE.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Shen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Liang, Harley, Shen.

Acquisition of data. Fan, Liang, Qu, Harley, Shen.

Analysis and interpretation of data. Fan, Liang, Tang, Cui, Luo, Huang, Chen, Higgs, Jallal, Yao, Harley, Shen.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

We thank the patients, normal healthy volunteers, and rheumatologists at Shanghai Renji Hospital who participated in this study. We also thank Dr. Haibo Zhou and Wen Pan for their helpful comments.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES