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the condition for long periods, and some severely af fected patients are resistant to current treatment regimens1. TAO is an inflammatory autoimmune dis ease of the orbit. Signs and symptoms of TAO are caused by inflammation of orbital connective tissue, increased orbital volume due to overpro-duction of glycos aminoglycans, and enhanced adipogenesis. CD4+T cells are involved in the immune and proliferative responses, which play an important role in the occurrence and develop-ment of TAO2,3. At present, it is believed that the immune response of TAO is mediated mainly by CD4+T cells, and there is an imbalance between Helper T cells, Regulatory cells (Th1/Th2/Treg/Th17) four CD4+T cells and related cytokines3-5. MicroRNAs (miRNAs) are small single-stranded noncoding RNAs, about 21-25 nucleotides in len-gth6. They control gene expression by targeting the 3’,5’ UTR or the coding sequences of specif c mRNAs and triggering either translation repres-sion or RNA degradation. miR NAs have emer-ged as important modulators of immunity and cellular physiology6-10. miR-146a with multiple target genes is a typical multifunctional miRNA that is in the same cellular pathways according to different conditions, and it plays various life processes, characterized in terms of its regulatory role in immune regulation, cell proliferation, dif-ferentiation, apoptosis, and extracellular matrix metabolism. MiR-146a can be induced by PI3K /NF-kappa B pathway; in turn, miR-146a in PI3K /NF-kappa B pathway has multiple target genes, negative to the regulation of the immune respon-se11,12. CD4+T cells are involved in the immune and proliferative responses, which play an im-portant role in the occurrence and development

Abstract. – OBJECTIVE: The aim of the study was to explore the functional role of miR-146a in CD4+T cells of active thyroid-associated oph-thalmopathy (TAO) patients and to identify the possible molecular mechanism for the modula-tion of miR-146a in TAO.

PATIENTS AND METHODS: The experimen-tal group collected six cases of peripheral ve-nous blood of patients with active TAO. The healthy control group collected six cases of normal peripheral venous blood. All specimens excluded other eye diseases and autoimmune diseases, tumors, asthma, chronic inflamma-tion, Human Immunodeficiency Virus (HIV), re-cent history of trauma, infection, and showed normal thyroid function. All patients with active TAO and age- and sex-matched healthy control subjects in this study.

RESULTS: miR-146a is downregulated in active TAO CD4+T cells. NUMB was a target of miR-146a.

CONCLUSIONS: We observed that miR-146a expression was downregulated in CD4+T cells during the active stage of patients with TAO. At the same time, it was found that NUMB can be targeted by miR-146a in CD4+T cells in TAO pa-tients. Decreased microRNA-146a in CD4+T cells promotes ocular inflammation in active TAO by targeting NUMB.

Key Words:TAO, MicroRNA-146a, CD4+T cells, NUMB.

Introduction

Thyroid-associated ophthalmopathy (TAO) is the most common orbital disease. Despite sub-stantial new fndings1 in its cellular and molecular underpin nings, the pathogenesis of TAO remains unclear. Many patients with TAO have to endure

European Review for Medical and Pharmacological Sciences 2017; 21: 1803-1809

Z.-J. HU1, J.-F. HE1, K.-J. LI1, J. CHEN1, X.-R. XIE2

1Department of Ophthalmology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China2Department of Endocrinology, The Affiliated National Hospital of Guangxi, Medical University, Guangxi, Zhuang Region, China

Corresponding Author: Jian-Feng He, MD; e-mail: hejianf@163.com

Decreased microRNA-146a in CD4+T cells promote ocular inflammation in thyroid-associated ophthalmopathy by targeting NUMB

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of TAO. MiR-146a has some potential targets for the immune and proliferative response of TAO. A previous study13 showed low expression for miR-146a in TAO patients with orbital tissue and peri-pheral blood mononuclear cells (PBMC). The aim of this study was to explore the functional role of miR-146a in CD4+T cells of active TAO patients and to identify the possible molecular mechanism for the modulation of miR-146a in TAO.

Patients and Methods

PatientsDiagnoses of TAO and disease activity were

based on Bartley and Gorman criteria14. The experimental group collected six cases of peri-pheral venous blood of patients with active TAO (n = 6, four female and two male cases; mean ± SD age 30.16 ± 3.76 years). The healthy control group collected six cases of normal peripheral ve-nous blood (n = 6, three female and three male, mean ± SD age 27.66 ± 4.08 years). All specimens excluded other eye diseases and autoimmune di-seases, tumors, asthma, chronic inflammation, HIV, recent history of trauma, and infection, and normal thyroid function. All patients with active TAO and age- and sex-matched healthy control subjects were included in this study. The patien-ts with active TAO were collected from the First Affliated Hospital of Guangxi Medical Universi-ty’s Outpatient Department and Inpatient Ward. The healthy control subjects were recruited from among the graduate students and medical staff at the Guangxi Medical University. The study pro-tocol was approved by the Ethics Committee of Guangxi Medical University, and written infor-med consent was obtained from each patient be-fore the study started.

Cell Isolation and CultureA total of 120 ml of peripheral venous blood

was obtained from each subject and preserved in heparin. CD4+ T cells were isolated using human CD4+T cell enrichment cocktail (Stem Cell, Ca-talog #15062, Vancouver, BC, Canada). Human CD4+ T cells were maintained in immunocult-XF T cell expansion medium without the addition of xeno medium and serum in the medium (Stem Cell, Catalog #10982, Vancouver, BC, Canada).

Cell TransfectionThe CD4+T cells were seeded in six-well pla-

tes and cultured for 24 h before the transfection.

We transfected with miR-146a mimic and inhi-bitor (RiboBio, Guangzhou, Guangdong, China) using INTERFERin (Polyplus-transfection, Stra-sbourg, France) according to the manufacturer’s protocol. Cell samples were collected at 48 h after transfection for further analysis. Total RNA was extracted at 48 h after transfection. Reverse Tran-scription-Polymerase Chain Reaction (RT-PCR) was used to verify miR-146a expression.

Quantitative RT-PCRCD4+T cells were collected and lysed in

RNAiso for small RAN (TaKaRa, Dalian, China) for miRAN extraction using the One-Step pri-meScript miRNA cDNA Synthesis Kit (TaKaRa, Dalian, China). qPCR was conducted for reverse transcription using the ABI 7500 Sequence De-tection System (Applied Biosystems, Foster City, CA, USA) to detect the expression of 146a (Fw: 5’-TTA TTA AGT ATC CAG TGC AGG GTC CGA GG-3’and Rv: 5’-TTG CGG GAC ATC TAA TAC TGC CTG GTA ATG-3’) (TaKaRa, Dalian, Chi-na), standardized by U6 reference (Fw: 5’-GGA ACG ATA CAG AGA AGA TTA GC-3’and Rv5’-TGG AAC GCT TCA CGA ATT TGC G-3’) (Ta-KaRa, Dalian, China). Detection was conducted in triplicate for each sample and data were calcu-lated with the 2-ΔΔCt method.

MiRNA Target PredictionBioinformatics methods were applied for the

prediction of targeted genes of miR-146a. The bioinformatics algorithm from TargetscanHuman (http://www.targetscan.org) was used.

Plasmid Construction and Double Luciferase Reporter Assay

To verify whether NUMB directly binds to miR-146a, a dual Luciferase assay was perfor-med. The 3’UTR of NUMB was amplifed by polymerase chain reaction (PCR) genomic DNA. The production was inserted into the downstream of NUMB 3’UTR reporter plasmids (Prl-numb) (Promega, Madison, WI, USA). The whole pla-smid was verifed by sequencing. The mutation in the miR-146a binding site of the NUMB 3’UTR was constructed by the GenePharma Company (GenePharma, Shanghai, China). The Luciferase reporter-containing mutant was constructed. For Luciferase assays, 293 T cells were transfected with Luciferase reporter plasmid along with miR-146a mimics or negative control by Lipofectami-ne2000 (Invitrogen, Carlsbad, CA, USA). At 48 h after transfection, these cells were analyzed by

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using a dual Luciferase assay kit (Promega, Ma-dison, WI, USA).

Western BlotAll reagents were prepared according to ma-

nufacturer’s recommendations for Wes (Pro-teinSimple, San Jose, CA, USA). Total protein of cells was extracted using radioimmunopreci-pitation assay (RIPA) protein extract (Beyotime Biotechnology, Shanghai, China). The extracted protein was quantifed using bicinchoninic acid (BCA) protein quantitative detection kit (Ther-mo-Fisher Scientifc, Waltham, MA, USA). Sam-ples were diluted to adjust protein concentration. The fnal samples of 5 ml each were boiled for 5 min, placed on ice for 5 min, briefly centrifuged, and then applied to proper wells. The Wes is ca-pable of performing loading, separation, washing, blocking, and immune-detection. After plate loa-ding, the separation of proteins by electrophoresis and immune-detection took place in the capillary system and were fully automated. Sample We-stern analysis was carried out at room temperatu-re and instrument default settings. NUMB (1:50 dilution) primary antibodies (ab177465, Abcam, Cambridge, MA, USA) were diluted with antibo-dy diluent (ProteinSimple, San Jose, CA, USA). The digital images were analyzed with Compass software (ProteinSimple, San Jose, CA, USA) on Wes. Band density differences were expressed as

percent of values. Data were collected from three independent experiments.

Statistical AnalysisData are expressed as the mean ± standard

(SD). The Mann-Whitney U-test was used to compare the values of miR-146a between the two groups. The comparison among other groups was estimated by the Student’s t-test. All results were analyzed using SPSS17.0 (SPSS Inc., Chicago, IL, USA) and p<0.05 was considered signifcant.

Results

miR-146a is Downregulated in TAO CD4+T cells

In this study, the expression level of miR-146a was detected by qPCR, and we observed that miR-146a was signifcantly downregulated in active TAO CD4+T cells compared with the con-trol (Figure 1).

NUMB was a Target of miR-146a By using the database of TargetscanHuman

(http://www.targetscan.org) to predict the target gene of miR-146a, we selected the potential target genes of NUMB as miR146a. To further confrm the direct target gene of miR146a for NUMB, we constructed a reporter gene expression vec-tor of the NUMB gene. The 3’-UTR fragment of the NUMB gene was amplifed by the PCR method, and then, the fragment was inserted into the expression vector to carry out sequencing to confrm that the vector was constructed correctly. A dual luciferase reporter gene assay system was used to demonstrate that NUMB is a direct target of miR-146a. The results confrmed that NUMB was a validated target of miR-146a (Figure 2).

Expression Changes of miR-146a and NUMB protein in CD4+T cell After Transfection

To assess whether miR-146a directly regula-tes NUMB, we transfected primary CD4+T cells from healthy subjects and patients with a mimic of miR-146a, an inhibitor of miR-146a, blank con-trol, or negative control. qRT-PCR results showed that the miR-146a mimic group had a higher expression level of miR-146a than the negative control (NC) and the blank groups (p<0.05). The miR-146a inhibitor group had a lower expres-sion level of miR-146a compared to the NC and the blank groups (p < 0.05). There was no diffe-

Figure 1. miR-146a is expressed at lower levels in CD4+T cells from patients with active TAO compared to normal control subjects. The miR-146a levels in CD4+T cells from TAO and matching adjacent normal control subjects were tested by qRT-PCR. The qRT-PCR experiments were per-formed three times. Data are mean ± SD *p<0.05.

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rence shown in the expression level of miR-146a between the NC group and blank group (p>0.05) (Figures 3A-B). Moreover, decreased levels of the NUMB protein were found in cells transfected with a miR-146a mimic relative to the NC and the blank groups (p<0.05), whereas increased le-vels of the NUMB protein were observed in cells transfected with a miR-146a inhibitor relative to the NC and the blank groups (p<0.05). There was no difference shown in levels of NUMB protein between the NC group and blank group (p>0.05) (Figure 3C-D). At the same time, we investiga-ted the mRNA expression level of NUMB, and

reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that neither miR-146a mimic nor miR-146a inhibitor influenced the expression of NUMB (p>0.05) (Figures 3E-F).

Expression of NUMB Then, we detected the NUMB protein levels in

the CD4+T cells and found the mean expression of NUMB in active TAO patients was higher than that in healthy subjects (p<0.05) (Figure 4).

Discussion

In this work, we observed that miR-146a expression was downregulated in CD4+T cells in the active stage of TAO patients. At the same time, we found that NUMB can be targeted by miR-146a in CD4+Tcells in TAO patients. NUMB15, a cell-fate determinant, is a tyrosine-binding do-main-containing protein that is linked to the cell’s membrane. It is one of the most important cell-fa-te determinants in living organisms. NUMB also is widely distributed in mature tissues, widely involved in the balance of differentiation/prolife-ration, regulation of apoptosis, cell migration, tis-sue regeneration, and other important physiologi-cal processes. Uemura et al15 have discovered and identifed a new gene in the study of the embryonic tissue of the fruit fly. NUMB is considered essen-tial for the formation of the sensory organ of Dro-sophila to determine the cell differentiation fate. Subsequent researches have found that the loss of NUMB function will lead to the loss of nerve cel-ls in the sensory organ development15. In contrast, overexpression of NUMB leads to abnormal dif-ferentiation, which makes the original supporting cells into neural cells, thus determining the role of NUMB as an endogenous cell fate determinant in the asymmetric cell division16,17. Previous studies have demonstrated that miR-146a is implicated in cell apoptosis. It was reported that upregulation of miR-146a induces apoptosis of human chon-drocytes18 and miR-146a regulated the maturation and differentiation of vascular smooth muscle cel-ls19. Also miR-146a induces apoptosis in several types of cancer20, including non-small cell lung cancer21, gastric cancer19, and breast cancer20. Mo-reover, miR-146a has been found to participate in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythemato-sus, and multiple sclerosis22-24. A recent work has shown that aberrant miRNA expression is asso-ciated with the pathogenesis of many diseases,

A NUMB-F 5’ CTTTATTTTTATGCATTTA-

GAATATTTTAAATagttctcAGATATTAAGA-AGTTGTATGA3’

| | | | | | |

NUMB-R 3’ CTTTATTTTTATGCATTTA-GAATATTTTAAATctacacaAGATATTAAGA-AGTTGTATGA 5’

B

Figure 2. NUMB may be a target gene of miR-146a (A, the 3’UTR sequence of NUMB was matched by detecting miR-146a with TargetScan software; B, a dual Luciferase repor-ter system was applied for testing the gene activity. Tran-sfection of miR-146a and mutant NUMB mRNA 3’UTR expression vector group, the relative activity of Luciferase was decreased signifcantly in transfection of miR-146a and wild type NUMB mRNA3’UTR expression vector group (p<0.05), while no obvious change of the relative activity of Luciferase was discovered in the transfection of miR-146a and mutant NUMB mRNA3’UTR expression vector group (p>0.05). *p<0.05.

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Figure 3. The expression levels of miR-146a were analyzed after transfection with miR-146a mimic or inhibitor, negative control, and blank. NUMB expression in CD4+T cells compared with the blank control group and negative control group. (A) After transfection miR-146a mimics or inhibitor, negative control, blank, the miR-146a levels in CD4+Tcells from patients with active TAO were examined. (B) After transfection miR-146a mimics or inhibitor, negative control, blank, the miR-146a levels in CD4+Tcells from healthy control subjects were examined. (C) MiR-146a mimics or inhibitor was transfected into CD4+Tcells. At 48 h later, the NUMB protein level in CD4+T cells from patients with active TAO tested by Western blot. (D) MiR-146a mimics or inhibitor was transfected into CD4+Tcells. At 48h later, the NUMB protein level in CD4+T cells from healthy control subjects tested by Western blot. (E) The relative gene expression levels of NUMB from patients with active TAO. (F) The relative gene expression levels of NUMB from healthy control subjects. *p<0.05.

A B

C D

E F

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such as tumors25. miR-146a with multiple target genes is a typical multifunctional mRNA that is in the same cellular pathways according to diffe-rent conditions, and it plays various life processes, characterized in terms of their regulatory role in immune regulation, cell proliferation, differentia-tion, apoptosis, and extracellular matrix metabo-lism26. Further studies were warranted to explore how miR-146a targets certain genes in CD4+T cells with inactive TAO.

Conclusions

We not only observed the downregulation of miR-146a expression in CD4+T cells of active TAO patients, but also verifed that miR-146a directly regulates the protein expression level of NUMB. We hope that this investigation provides assistance to explore further the pathogenesis of miR-146a in active TAO and in developing new treatment methods.

AcknowledgmentsThis work is supported by grants from the National Natural Science Foundation of China (No.81360152).

Conflict of interestThe authors declare no conflicts of interest.

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