Down-regulation of microRNA-451a facilitates the activation and proliferation of CD4+ T cells by targeting Myc in patients with dilated cardiomyopathy

CD4+ T cells are abnormally activated in patients with dilated cardiomyopathy (DCM) and might be associated with the immunopathogenesis of the disease. However, the underlying mechanisms of CD4+ T cell activation remain largely undefined. Our aim was to investigate whether the dysregulation of microRNAs (miRNAs) was associated with CD4+ T cell activation in DCM. CD4+ T cells from DCM patients showed increased expression levels of CD25 and CD69 and enhanced proliferation in response to anti-CD3/28, indicating an activated state. miRNA profiling analysis of magnetically sorted CD4+ T cells revealed a distinct pattern of miRNA expression in CD4+ T cells from DCM patients compared with controls. The level of miRNA-451a (miR-451a) was significantly decreased in the CD4+ T cells of DCM patients compared with that of the controls. The transfection of T cells with an miR-451a mimic inhibited their activation and proliferation, whereas an miR-451a inhibitor produced the opposite effects. Myc was directly inhibited by miR-451a via interaction with its 3′-UTR, thus identifying it as an miR-451a target in T cells. The knockdown of Myc suppressed the activation and proliferation of T cells, and the expression of Myc was significantly up-regulated at the mRNA level in CD4+ T cells from patients with DCM. A strong inverse correlation was observed between the Myc mRNA expression and miR-451a transcription level. Our data suggest that the down-regulation of miR-451a contributes to the activation and proliferation of CD4+ T cells by targeting the transcription factor Myc in DCM patients and may contribute to the immunopathogenesis of DCM.

Dilated cardiomyopathy (DCM), 3 characterized by a left ventricular dilation and systolic dysfunction, is the most common form of cardiomyopathy and ranks as one of the paramount conditions leading to congestive heart failure and thus heart transplantation (1,2). Systemic immune activation and chronic inflammation are involved in the development and progression of DCM. Several lines of evidence have suggested that CD4 ϩ T cells play a critical role in the inflammation of DCM. Circulating CD4 ϩ T cells in patients with DCM exhibit high levels of expression of the surface activation markers CD25, CD69, HLA-DR, and CD40L (3,4). CD4 ϩ T cells are found to recruit the endomyocardial biopsy site and release pro-inflammatory cytokines (e.g. tumor necrosis factor-␣, interleukin-18, and interferon-␥), which contribute to myocardial apoptosis and fibrosis (5). CD4 ϩ T cells are indispensable for B cells to become activated and to secrete high affinity antibodies. Several autoantibodies against cardiac proteins (e.g. myosin, troponin I, and ␤1-adrenergic receptors) have been detected in sera from patients with DCM (6 -8). These autoantibodies, belonging to IgG class 3, are associated with the poor development of left ventricular function and can predict sudden death in DCM patients (6,8).
MicroRNAs (miRNAs) are a class of small (ϳ21 nucleotides in length) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level, typically by binding to their targeted sites located in the 3Ј-untranslated region (3Ј-UTR) of mRNAs. miRNAs participate in the process of T lymphocyte development, differentiation, activation, and aging (9). Overexpression of miRNA-155 (miR-155) in patients with atopic dermatitis promotes T cell activation and proliferation via the direct targeting of cytotoxic T lymphocyte-associated antigen 4 (10). Up-regulation of miR-126 modulates the DNA methylation by directly targeting DNA methyltransferase 1, contributing to T cell autoreactivity in systemic lupus erythematosus patients (11). However, to the best of our knowledge, the miRNA expression profile and its relationship with CD4 ϩ T cell activation in DCM patients remain unclear.
In this study, we first uncovered that CD4 ϩ T cells from DCM patients showed increased expression of the surface activation markers CD25 and CD69 and enhanced proliferation in response to anti-CD3/28. Furthermore, we demonstrated that the miRNA expression profiles of CD4 ϩ T cells derived from DCM patients exhibited significant differences from those of the controls. As miR-451a is associated with the activation of CD4 ϩ T cells in other studies (12,13), we focused on the role of miR-451a in DCM. The results revealed that in patients with DCM, miR-451a facilitated the activation and proliferation of CD4 ϩ T cells by targeting Myc.

Increased expression levels of surface activation markers and enhanced proliferation in CD4 ؉ T cells of DCM patients
We first analyzed the expression levels of surface activation markers, including CD25, CD69, and MHC-II on CD4 ϩ T cells and their proliferation in response to anti-CD3/28 stimulation by flow cytometry. As shown in Fig. 1, A and B, the expression of the early activation markers CD25 and CD69 on CD4 ϩ T cells was significantly increased in the DCM patients compared with the controls (CD25, 10.3 Ϯ 3.9% versus 5.0 Ϯ 1.8%, p Ͻ 0.05; CD69, 5.7 Ϯ 1.9% versus 2.8 Ϯ 1.9%, p Ͻ 0.05), whereas the expression of the late activation marker MHC-II on CD4 ϩ T cells showed a non-significant trend toward an increase in the DCM patients (8.3 Ϯ 3.6% versus 6.1 Ϯ 3.2%; p ϭ 0.14). To measure T cell proliferation, the cells were stimulated with anti-CD3/28, and the division index was calculated using the proliferation platform. As shown in Fig. 1, C and D, the division index of the CD4 ϩ T cells from the DCM patients was significantly higher than that of the controls (1.68 Ϯ 0.11 versus 1.30 Ϯ 0.10; p Ͻ 0.01). Our data indicate that the CD4 ϩ T cells are abnormally activated in DCM patients.

Altered miRNA profile and decreased miR-451a expression in CD4 ؉ T cells of DCM patients
To identify whether there is a correlation between miRNAs and abnormal activation of CD4 ϩ T cells in DCM, we per- Figure 1. Expression of surface activation markers, including CD25 and CD69, and cell proliferation in response to the anti-CD3/28 stimulation increase in circulating CD4 ؉ T cells of DCM patients. A, representative FACS images from one DCM patient and one control subject are shown, indicating CD25, CD69, and MHC-II expression levels in gated CD4 ϩ T cells. B, frequencies of CD25 ϩ , CD69 ϩ , and MHC-II ϩ cells in circulating CD4 ϩ T cells were compared between DCM patients (n ϭ 12) and controls (n ϭ 11). C, PBMCs were labeled with CFSE and then stimulated with anti-CD3/28 for 3 days as indicated under "Experimental Procedures." The proliferation was determined using gated CD4 ϩ T cells by FACS. D, proliferation of CD4 ϩ T cells, calculated as a division index using the FlowJo proliferation platform, was compared between the DCM patients (n ϭ 12) and controls (n ϭ 11). *, p Ͻ 0.05, and **, p Ͻ 0.01 versus control group.
As shown in Table 1, the study population in the validation groups was different in incidence of diabetes and their use of ACEI/ARBs, ␤-blockers, diuretics, or digitalis. To adjust the potential confounding effect on miRNA-451a expression for the above variables, we performed a multiple linear regression analysis. The results indicated that diabetes or medical therapies did not influence miRNA-451a expression in CD4 ϩ T cells ( Table 2).

Role of miR-451a in the activation and proliferation of T cells
To explore the potential role of miR-451a in the activation and proliferation of T cells, we up-regulated and down-regulated miR-451a in Jurkat T cells by transfecting cells with an miR451a mimic and an miR451a inhibitor, respectively. The efficiency of the miR-451a mimic and inhibitor transfection was determined by RT-PCR; the results demonstrated that the miR-451a expression levels were increased 3.56-fold in the miR-451a mimic group and decreased to 29.1% in the miR-451a inhibitor group compared with the negative control (NC) groups, respectively (Fig. 3A). The transfection of Jurkat T cells with the miR-451a mimic resulted in marked decreases in CD25 and CD69 expression, whereas the miR-451a inhibitor greatly increased the expression of these activation markers (Fig. 3, B and C). We also determined the effect of miR-451a on the proliferation of and IL-2 production by T cells. Both gainof-function and loss-of-function experiments demonstrated a suppressive effect of miR-451a on the proliferation of and IL-2 production by Jurkat T cells (Fig. 3, D and E). Collectively, these data suggest that miR-451a regulates the activation and proliferation of T cells.

Identification of Myc as a target of miR-451a in T cells
Next, we investigated the target of miR-451a that might modulate T cell activation and proliferation. The T cell activation-associated transcription factor Myc is a predicted target of miR-451a according to the mirTarBase database (14). To verify this possibility, we generated a firefly luciferase reporter vector fused downstream to a segment of the Myc 3Ј-UTR containing either the wild-type putative miR-451a-binding sequence (Myc-3Ј-UTR-WT) or a point mutant putative miR-451a-binding sequence (Myc-3Ј-UTR-MUT) (Fig. 4A). The constructs were then co-transfected into Jurkat T cells with an miR-451a mimic or an miRNA NC, and the luciferase activity was mea-sured 48 h later. Compared with miRNA NC, miR-451a specifically suppressed Myc-3Ј-UTR-WT luciferase activity but failed to inhibit Myc-3Ј-UTR-MUT luciferase activity (Fig. 4B).
We also validated the regulation of Myc by miR-451a using RT-PCR and western blotting. The transfection of Jurkat T cells with an miR-451a mimic led to a dramatic decrease in Myc expression, whereas an miR-451a inhibitor increased Myc expression at both the mRNA and protein levels ( Fig. 4, C and D).

Role of Myc in the activation and proliferation of T cells
To further investigate the role of Myc in T cell activation and proliferation, we knocked down Myc in Jurkat T cells using Myc-specific siRNA. Myc expression was effectively decreased by siRNA at both the mRNA and protein levels in Jurkat T cells (Fig. 5, A and B). Myc knockdown dramatically decreased the expression of surface activation markers on Jurkat T cells (

Correlation of decreased miR-451a expression and increased Myc expression in CD4 ؉ T cells from DCM patients
Given that our results indicated that miR-451a regulates Myc in the T cells, we next measured the expression of Myc in CD4 ϩ T cells by RT-PCR. As shown in Fig. 6A, the expression levels of Myc were significantly increased in CD4 ϩ T cells from DCM patients compared with those from controls. In addition, we plotted the expression of miR-451a in DCM patients against the expression of Myc from the same samples. A strong inverse correlation was observed between the two values (r ϭ Ϫ0.527, p Ͻ 0.05, Fig. 6B). Taken together, these data indicate that the up-regulation of Myc by miR-451a promotes CD4 ϩ T cell activation and proliferation in DCM.

Discussion
Increasing studies have uncovered the involvements of miRNAs in the pathogenesis of DCM. A distinct miRNA profile was revealed in the failing hearts of DCM patients, and miR-340 was identified as a key miRNA in regulating the structure of cardiomyocytes (15). A low let-7i level was found in endomyo- Table 1 Clinical characteristics of the study population Data are presented as mean Ϯ S.D., percentages, or numbers. NYHA, New York Heart Association functional class; LVEF, left ventricular ejection fraction; LVEDD, left ventricular end diastolic diameter; NT-pro-BNP, N-terminal brain natriuretic peptide; ACEI/ARBs, angiotensin-converting-enzyme inhibitors/angiotensin receptor blockers.    (17,18). Apart from a direct role in the heart, miRNAs seem to be altered and to regulate gene expression in other tissues or organs, serving as another mechanism to participate in DCM. The levels of miRNA-548c were reduced in circulating peripheral blood mononuclear cells (PBMCs) from DCM patients and could be used as a reliable biomarker to predict cardiac dysfunction (19). The plasma concentration of miR-423-5p was elevated and was positively correlated with the level of N-terminal brain natriuretic peptide, thus serving as a diagnostic biomarker for heart failure caused by DCM (20). The immune inflammation mediated by CD4 ϩ T cells is implicated in the development of DCM (21). However, the role of miRNAs in the aberrant activation of CD4 ϩ T cells associated with DCM remains poorly understood.

Characteristics
In this study, we first revealed a distinct pattern of miRNA expression in CD4 ϩ T cells from DCM patients compared with controls, indicating that miRNAs may be involved in the aberrant T cell activation observed in DCM patients. Second, we found that miR-451a was markedly down-regulated in CD4 ϩ T cells of DCM patients and regulated cell activation and prolif-eration. Previous studies revealed that some medications for treating heart failure, such as ACEI or ␤-blockers, could influence miRNA expression (22)(23)(24). In our study, we found that down-regulation of miR-451a in CD4 ϩ T cells is independently associated with DCM and is not influenced by medical treatment.
Currently, the role of miR-451a in cardiovascular diseases has not been well characterized. Recent studies show that miR-451a is involved in modulation of myocardial lipid accumulation and ischemia-induced myocyte death. Ono and co-workers (25) have found that miR-451a exacerbates lipotoxicity and cardiac hypertrophy in high fat diet-induced diabetic cardiomyopathy. miR-144 and miR-451, two conserved miRNAs, are processed from a single gene locus through the regulation of the transcription factor GATA-1 (26). Fan and co-workers (27) uncovered that the overexpression of the miR-144/451 cluster conferred protection against ischemia/reperfusion-induced cardiomyocyte death in adult rat ventricular myocytes by targeting the CUGBP2-COX-2 pathway, and the down-regulation of miR-144/451 exhibited the opposite effects. Furthermore, loss of the miR-144/451 cluster impaired the ischemic preconditioning-induced protective effects in the mouse heart (28). In our study, the expression level of miR-144 was not significantly different in the circulating CD4 ϩ T cells of the DCM and the controls groups (data not shown). These results may be due to   the expression levels of mature miR-144 and miR-451 exhibiting specificity in different cells, because the processing pathway of miR-451 requires Ago2 slicer catalytic activity, not Dicer (29).
Myc is a pleiotropic transcription factor that regulates a variety of cellular processes, including cell cycle progression, metabolism, cell growth, and apoptosis (30). Previous studies show that Myc could be induced by T cell antigen receptor (TCR) signal and various cytokines in T cells and is involved in cell activation and proliferation by regulating the genes responsible for metabolic reprogramming and cell cycle (31)(32)(33). In agreement with these findings, we found that inhibition of Myc blocked the TCR agonist-induced T cell activation and proliferation. IL-2 has been reported to be a critical regulator of Myc expression at the post-transcriptional level (34). Our data show that inhibition of Myc prevents activated T cells to secret IL-2, suggesting that there may be a regulatory loop between IL-2 and Myc. It has been reported that miR-451a regulates the growth of tumors such as acute lymphoblast leukemia, head and neck squamous cell carcinomas, and lung adenocarcinoma by directly targeting Myc (35)(36)(37). Here, we show that miR-451a directly targets Myc in T cells in vitro, and their expressions are negatively correlated in CD4 ϩ T cells of DCM patients in vivo, probably suggesting a role of miR-451-Myc pathway in CD4 ϩ T cell abnormality in DCM conditions. Still, there are limitations to this study. First, the sample size of our study population is relatively small. Second, the mechanisms underlying the down-regulation of miR-451 are not investigated in this study and deserve further study.
In conclusion, we found that the expression of miR-451a is down-regulated in CD4 ϩ T cells from DCM patients and that this down-regulation contributes to increasing Myc expression and inducing the aberrant T cell activation in DCM. This study sheds new light on the regulation of Myc by miRNAs and suggests that miR-451a may be a promising therapeutic intervention to treat chronic systemic inflammatory diseases such as DCM.

Study population
A total of 50 patients with DCM without detectable etiology were recruited from an in-patient ward at the Union Hospital, Huazhong University of Science and Technology. The diagnosis of DCM was based on the guidelines of the European Society of Cardiology Working Group on the classification of cardiomyopathies (38). A left ventricular end diastolic diameter Ͼ5.5 cm and left ventricular ejection fraction Ͻ45%, as assessed by echocardiography, were used as inclusion criteria for the recruitment of patients. The exclusion criteria included the presence of coronary heart disease confirmed by coronary angiography, hypertensive heart disease, valvular heart disease, other autoimmune diseases, serious infection, pregnancy, endocrine disease, tumors, or receiving immunosuppressive agents (glucocorticosteroid). Peripheral blood samples were collected into heparinized tubes. Samples of five randomly selected DCM patients were assigned for an miRNA screening assay, with samples of five age-and sex-matched controls used for comparison. Samples from the remaining 45 DCM patients and 45 normal controls were used for validation tests. The clinical characteristics of the study population are listed in Table 1. The investigation conforms to the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee of Tongji Medical College of Huazhong Science and Technology University, and written informed consent was obtained from all participants.

Isolation of PBMCs and purification of CD4 ؉ T cells
PBMCs were isolated from freshly heparinized blood using Ficoll-Histopaque (Sigma), and CD4 ϩ T cells were purified from PBMCs by MACS using a human CD4 ϩ T cell isolation kit (Miltenyi Biotec, Germany) following the manufacturer's protocols (39). The purity of the CD4 ϩ T cells was Ͼ97%.

Cell cultures
Jurkat T cells (human peripheral blood leukemia T cells, E6.1) were purchased from ATCC (American Type Culture Collection). The Jurkat T cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco Life Technologies, Inc.), 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO 2 as described previously (40).

miRNA microarray profilings
Total RNA of CD4 ϩ T cells was extracted using TRIzol reagent (TaKaRa, China) according to the manufacturer's instructions, and the freshly extracted total RNA was stored at Ϫ80°C for subsequent testing. A total of 1 g of total RNA was used for the expression analysis with GeneChip miRNA 4.1 arrays (Affymetrix), which includes 2578 human mature miRNAs based on miRBase version 20. Briefly, the total RNA was labeled with biotin using a FlashTag biotin RNA labeling kit according to the manufacturer's protocol and was subsequently hybridized overnight. The miRNA chips were washed and stained using the Affymetrix GeneChip hybridization wash and stain kit and were then scanned with an Affymetrix GeneChip Scanner 3000. The CEL-files of the raw data were extracted by Affymetrix GeneChip Command Console Software. The differential expression between the two groups was considered as significant if the fold changes are greater than 1.5 between the two groups using an independent t test by R statistical software.

Western blotting
Protein was extracted from Jurkat cells in RIPA buffer with a protease inhibitor mixture (Roche Applied Science) and separated on 10% SDS-PAGE (Boster, China) to quantify the level of Myc. GAPDH was used as an endogenous control. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), and the blots were incubated with the appropriate antibodies, anti-Myc (1:1000; Cell Signaling Technology) and anti-GAPDH (1:10,000; Abcam), followed by incubation with the HRP-conjugated secondary goat anti-rabbit (1:10,000; Boster, China). The signals were visualized using enhanced chemiluminescence substrates (Pierce).

Dual-luciferase reporter assay
Jurkat T cells were cultured in 24-well plates at ϳ5 ϫ 10 4 cells/well and transfected with 200 ng of either pMIR-RE-PORT-Myc-3ЈUTR-WT or pMIR-REPORT-Myc-3ЈUTR-MUT and 100 nM miR-451a mimic or negative control NC-miRNA mimics, along with 20 ng of Renilla luciferase vector for normalization as described previously (42). The cell extracts were prepared 48 h after transfection, and the luciferase activity was measured using a Dual-Glo TM luciferase assay system (E2920, Promega).

ELISA
After 48 h, the transfected Jurkat T cells were stimulated with 5 g/ml ␣CD3 mAb and 2.5 g/ml ␣CD28 mAb as described above, and the culture supernatants were collected 24 h after stimulation to detect the IL-2 concentration using an IL-2 ELISA kit (Elabscience, China) according to the manufacturer's instructions.

Cell counting kit-8 (CCK-8) assay
CCK-8 assay was performed to assess the proliferation of Jurkat T cells. After 48 h of transfection, the Jurkat T cells were seeded in 96-well plates in RPMI 1640 medium containing 10% FBS at 1 ϫ 10 4 cells/well and stimulated with anti-CD3/CD28 mAb for 24 h. Then, 10 l of CCK-8 solution (Dojindo Laboratories, Japan) was added to each well, and the samples were incubated for 4 h at 37°C. The absorbance at 450 nm was measured using a microplate reader, and the relative proliferation index was measured by the absorbance value of the treatment compared with that of the control. All samples were measured in triplicate.

Statistical analysis
The data are expressed as the mean Ϯ S.D. Student's t test was used to analyze pairs of continuous variables, and the 2 test was used for categorical variables. Two-sided Spearman's correlation test was used to calculate the correlation between the relative expression levels of miR-451a and Myc. To exclude the influence of potential confounders to miR-451a expression, we adjusted for diabetes and medications using a multiple linear regression model. p Ͻ 0.05 was considered to be significant.