ABSTRACT The transcription factor FOXP3 is essential for the differentiation and function of regulatory T cells (Treg). It is established that the transcription factor GATA-3 is induced in

Treg cells under inflammatory conditions. GATA-3 stabilizes FOXP3 levels to avoid the differentiation of Treg cells into inflammatory-like T cells. The IL-6 signal pathway influences the

sensitivity of Treg cells towards instability. The mechanism of GATA-3 in regulating FOXP3 and its relation to the IL-6 pathway remains unclear. Here we report how miR-125a-5p plays an

important role in regulating the conversion of Treg cells by IL-6. miR-125a-5p expression is low in Treg cells under steady state conditions and can be induced by GATA-3 to inhibit the

2021 MIR-146A REGULATES REGULATORY T CELLS TO SUPPRESS HEART TRANSPLANT REJECTION IN MICE Article Open access 17 June 2021 THE MIR-641-STIM1 AND SATB1 AXES PLAY IMPORTANT ROLES IN THE

REGULATION OF THE TH17/TREG BALANCE IN ITP Article Open access 16 May 2024 INTRODUCTION Treg cells maintain the balance of immune self-tolerance and homeostasis via limiting aberrant or

excessive inflammation1,2. Treg cells are not terminally differentiated cells, as they can lose the expression of FOXP3 and become pro-inflammatory cells when induced by certain

cytokines3,4,5. IL-6 is one of the most likely candidates to induce FOXP3 downregulation as it plays a critical role in determining the balance between Th17 and Treg differentiation6. In

year 2003, Pasare and Medzhitov reported that Toll-like receptor-induced IL-6 expression led to the loss of Treg cell function7. More recently, activated Treg cells were found to

differentiate into Th17 cells in the presence of IL-6 and in the absence of exogenous TGF-β8. IL-6, together with IL-1, may induce downregulation of Foxp3 in a signal transducer and

activator of transcription 3 (STAT3)-dependent pathway9. _In vivo_ experiments have also shown that in autoimmune arthritis FOXP3+ Treg cells lose FOXP3 expression and undergo conversion

into Th17 cells. This process was found dependent on IL-610. It has been reported that IL-6 downregulates FOXP3 mRNA expression via epigenetic changes11. Furthermore, our laboratory has

shown that IL-6 and TGF-β treatment can downregulate FOXP3 by promoting FOXP3 protein degradation _in vitro_12. However, how the IL-6 signal pathway is regulated in Treg cells remains

unclear. MicroRNAs (miRNAs) have emerged as important regulators in many physiological and pathological processes including development, differentiation, metabolism, immunity, cell

proliferation and apoptosis13,14. miRNAs are small non-coding RNAs, which repress translation or cleave messenger RNAs (mRNAs) via binding to the coding sequence (CDS) regions or the 3′

untranslated regions (3′UTR) of target genes. For instance, miR-146a has been reported as indispensable for Treg-mediated suppression through regulating the IFNγ response by targeting

STAT115. FOXP3 directly induces miR-155 and is responsible for the survival of Treg cells through repression of suppressor of cytokine signaling 1 (SOCS1)16,17. All these findings suggest

that miRNAs play important roles in the function of Treg cells. In this study, we performed a miRNA microarray and Real-Time-PCR analysis on a human Treg-like cell line to correlate miRNA

expression with FOXP3 expression. We identified that miR-125a-5p, a GATA3-inducible miRNA, targets interleukin 6 receptor (IL-6R) and STAT3 transcripts. We propose a regulatory mechanism by

which miR-125a-5p reduces the sensitivity of Treg cells toward IL-6 conversion. RESULTS MIR-125A-5P EXPRESSION IS REGULATED IN HUMAN T CELL LINES AND T SUBSETS FOXP3 is the master

transcription factor in Treg cells, which regulates the function of Treg cells via modulating gene transcription. We were interested in knowing whether any miRNAs were related to FOXP3

expression. The SZ-4 cell line was originally identified in a Sezary disease patient and expresses FOXP318. This cell line has been used to investigate Sezary Syndrome and the regulation of

genes in T cells19. We purified RNA from the FOXP3-positive SZ-4 cell line and FOXP3-negative SZ-4 cell line (derived from subcloning) and carried out miRNA microarray to compare expression

levels of miRNA. The clusters of miRNAs that had more than two-fold change in expression were rendered into a heat-map diagram (Fig. 1A). miR-125-5p was one of the most downregulated miRNAs

revealed by the microarray data. We then used Real-Time-PCR to confirm the expression levels of several miRNAs in the SZ-4 (Fig. 1B) and Jurkat (Fig. 1C) cell lines. All of these results

indicated that miR-125a-5p is downregulated in FOXP3-positive T cells. We then checked the expression levels of miR-125a-5p in primary Th0, Th1, Th2, Th17, iTreg and nTreg cells. The

expression of miR-125a-5p was found lower in resting nTreg cells compared with effector T cells, but could be upregulated after TCR activation (Fig. 1D). GATA3 INDUCES THE EXPRESSION OF

MIR-125A-5P IN TREG CELLS In order to know whether the expression of miR-125a-5p is regulated by FOXP3, we analyzed the promoter region of miR-125a-5p for transcription factor binding DNA

epitopes. There were no predicted binding sites for FOXP3, but rather those for GATA3 binding were found (Supplemental Sequence 1). We then cloned the promoter region of the gene

transcribing miR-125a-5p into the pGL3-basic vector and carried out a luciferase assay in 293T cells (Fig. 2A). The data indicated that the activity of the miR-125a-5p promoter could be

induced by GATA3 in a dose dependent manner. Site-directed mutations suggested that site four was the most important binding site of GATA3 as luciferase activity after mutation of the fourth

site showed no change of luciferase activity with different levels of PIP-Myc-GATA3 (Fig. 2B). We then examined whether GATA3 could upregulate miR-125a-5p levels. GATA3 was overexpressed in

Jurkat and Jurkat-HA-FOXP3 cells by electroporation and we found the expression level of miR-125a-5p was upregulated (Fig. 2C). Endogenous GATA3 was then induced by anti-CD3/CD28 antibodies

in Jurkat-HA-FOXP3 cells and primary human nTreg cells. Protein levels of GATA3 was tested via Western blotting (Supplemental Fig. 1A) or flow cytometry (Supplemental Fig. 1B) and the

expression level of miR-125a-5p though Real-Time PCR (Fig. 2D). These results show that miR-125a-5p could be upregulated and this correlated with the endogenous induction of GATA3

expression. We then depleted GATA3 via lentivirus transduction in nTreg cells followed by anti-CD3/CD28 activation and detected GATA3 protein and miR-125a-5p levels. The results show that

the upregulation of miR-125a-5p in activated nTreg cells was dependent on endogenous GATA3 expression (Fig. 2E & Supplemental Fig. 1C). All the above data suggested that under TCR

stimulation the expression of miR-125a-5p could be upregulated via GATA3 in human nTreg cells. MIR-125A-5P DIRECTLY TARGETS IL-6R AND STAT3 In order to investigate the function of

miR-125a-5p we first identified its target genes. We searched for different target genes in TargetScan and then selected 42 target genes related to immune function. The 3′UTR of these genes

were cloned into the pGL3 vector for luciferase assays. We overexpressed or knocked down miR-125a-5p in 293T cells and analyzed for changes in luciferase activity. The results show that

miR-125a-5p significantly reduced luciferase activity using the 3′UTR of IL-6R and STAT3 but not after mutation of the miRNA interaction sites (MutIL6R-2 and MutSTAT3, respectively) (Fig.

3A,B). We then checked the protein levels of the target genes after overexpression or knockdown of miR-125a-5p in nTreg cells. Western blotting analysis showed that the protein level of

STAT3 was reduced by miR-125a-5p (Fig. 3C & Supplemental Fig. 2) and by flow cytometry IL-6R was found decreased by miR-125a-5p (Fig. 3D). The miRNA binding sites and mutations are shown

in Supplemental tables 1 and 2. MIR-125A-5P DECREASES THE SENSITIVITY OF TREG CELLS TOWARD IL-6 CONVERSION It is well known that IL-6 stimulation can reduce FOXP3 levels and decrease the

suppressive function of Treg cells, which is dependent on STAT3. As IL-6R and STAT3 are both targets of miR-125a-5p, we hypothesized that miR-125a-5p can influence the sensitivity of Treg

cells towards IL-6 signals. After overexpressing or knockdown of miR-125a-5p in primary nTreg cells, the cells were activated with anti-CD3/CD28 beads with or without IL-6 for 48 hours. The

expression level of FOXP3 in nTreg cells was then detected by flow cytometry. In the control groups (ago-NC and anti-NC), FOXP3 was downregulated when treated with IL-6. However, the

expression of FOXP3 had almost no change upon IL-6 treatment in the miR-125a-5p overexpression group and decreased significantly in the miR-125a-5p knockdown group (Fig. 4A). We also carried

out a suppression assay with the same group of nTreg cells. The suppressive functions of the four different groups of Treg cells were similar when treated only with anti-CD3/CD28 beads

(Fig. 4B). After IL-6 stimulation, the suppressive function of Treg cells decreased in all of the groups. Knockdown of miR-125a-5p highly sensitized the Treg cells to IL-6-mediated

downregulation of Treg suppression, but the overexpression of miR-125a-5p increased the suppressive function of these Treg cells under IL-6 stimulation (Fig. 4C). All these data suggest that

miR-125a-5p decreases the sensitivity of Treg cells toward IL-6-induced conversion. GATA3, MIR-125A-5P, IL-6R, STAT3 AND FOXP3 FORMS A REGULATORY LOOP As the decrease and dysfunction of

Treg cells has been found in some human diseases we tested whether miR-125a-5p expression correlated with disease occurrence. Our published work has already shown how GATA3 expression is

high in Treg cells of asthma patients20. Here, we investigated the expression level of miR-125a-5p and its target genes in asthma patients. The expression levels of GATA3 and miR-125a-5p

were significantly upregulated in the Treg cells of asthma patients compared with healthy donors (Fig. 5A,B). In addition, the expression level of IL-6R was significantly downregulated in

asthma patients (Fig. 5C). There was no difference in the expression of STAT3 between the asthma patients and healthy donors, albeit a cluster of patients did show relatively lower

expression of STAT3 in the asthma patients (Fig. 5D). The expression of FOXP3 was also upregulated (Fig. 5E). These results reveal how miR-125a-5p and IL-6R may play important roles in

asthma and show a regulatory pathway that regulates the sensitivity of Treg cells toward IL-6 conversion. DISCUSSION We investigated the role of miR-125a-5p in human Treg cells. mir-125a-5p

has been reported to act as a tumor suppression modulator in several kinds of cancers, such as glioblastoma, lung cancer and hepatocellular carcinoma etc.21,22,23,24,25 and has also been

reported to play a role in the immune system. In Drosophila, miR-125 was identified as a regulator of innate immunity26. In oxidized low-density lipoprotein-stimulated monocyte-derived

macrophages, miR-125a-5p was found to decrease lipid uptake and secretion of inflammatory cytokines27. mir-125a-5p was also reported to downregulate the expression of a hepatitis B virus

surface antigen28. During macrophage activation and polarization, miR-125a-5p promotes M2 macrophages and reduces M1 phenotype polarization29. However, the expression and function of

miR-125a-5p in Treg cells remained unclear. The expression of miR-125a-5p is lower in CD4+ CD25+ T cells compared to CD4+ CD25− cells purified from human cord blood30. Valproate treatment of

human cord blood CD4+ effector T cells could repress miR-125a-5p expression and upregulate FOXP331. In purified T cell subsets from PBMC of human, Real-Time-PCR results indicated that the

expression of miR-125a-5p is downregulated in Treg cells (resting) compared to effector T cells32. Our data also suggested that miR-125a-5p is expressed lower in FOXP3+ human T cell lines.

Many reports have indicated that miRNA expression can be regulated by transcription factors. The transcription factor Twist can induce miR-10b expression33 and HIF-1α can upregulate

miR-21034,35. Our data showed that TCR and IL-2 activation could upregulate miR-125a-5p and this process was dependent on GATA3. GATA3 is the canonical transcription factor of Th2

cells36,37. GATA3 is required for T cell lineage commitment as GATA3 is expressed in Linloc-KithiCD25− early T cell progenitors and lack of GATA3 results in failed development of T cells38.

GATA3 is indispensable for the early development of CD4+ T cells during the ß selection process39. It is reported that GATA3 binds to and promotes ThPOk to induce CD4+ and CD8+ T cell

differentiation40. In Treg cells, the expression of GATA3 is critical for Treg cell physiology during inflammation41. GATA3 is also reported as essential for the function of Treg cells via

binding to and promoting cis-acting elements of Foxp342. We found that GATA3 upregulates miR-125a-5p in Treg cells during TCR stimulation. But in Th2 cells whether GATA3 can up regulate

miR-125a-5p remains unknown. The expression of miR-125a-5p in resting Th1 and Th17 cells is higher than resting Treg cells, which may reflect other mechanisms that regulate miR-125a-5p in

other T cell subsets. In animals, miRNA sequences and their targets are not perfectly matched, which makes the search for targets complicated. Using a computer-based method, we identified

IL-6R and STAT3 as two novel targets of miR-125a-5p. IL-6 transduces two signal pathways via binding to IL-6R: one dependent on STAT3 activation and another on Src homology region 2

domain-containing phosphatase 2 (SHP2). It is reported that the expression level of IL-6R is responsible for the sensitivity of Treg cells toward IL-6 conversion43. IL-6-induced inhibition

of FOXP3 is dependent on STAT344. Also, STAT3 has been reported to promote the instability of Treg cells45. Thus we predicted that miR-125a-5p could regulate the sensitivity of Treg cells

under IL-6 stimulation and our _in vitro_ data had confirmed our prediction. Our data also showed that without IL-6 stimulation, overexpression or knock down of miR-125a-5p did not change

the expression level of FOXP3 or the suppressive function of Treg cells. Similar results have been reported that show how the overexpression of miR-125a have no effect on FOXP3 expression or

cell phenotype30. Fayyad-Kazan H _et al._ indicated that valproate treatment induces FOXP3 expression in CD4+ effector T cells by increasing the binding of Ets-1 and Ets-2 to the FOXP3

promoter, instead of a miR-125a-5p dependent mechanism31. It is probable that different cell types and experimental conditions led to these different observations. The IL-6 signal pathway is

also important for the differentiation of iTreg and Th17 cells. However, changes in the expression level of miR-125a-5p in naïve T cells had no effect on the polarization of both cell types

(data not shown). Also, the IL-6 signal pathway has been reported to be regulated by several miRNAs. In the malignant transformation of MCF-10A, Lin28 and let-7a regulate the activity of

the IL-6/STAT3 axis46. It is reported that there is one feedback loop comprised of IL-6-STAT3-miR-24/miR-629-HNF4-miR-124, which regulates hepatocellular oncogenesis47. miR-93 influences

proliferation and differentiation states of breast cancer stem cells (BCSCs) by targeting several genes including STAT348. All these studies were performed in cancer cells and whether these

miRNAs have similar roles to miR-125a-5p in Treg cells requires more investigation. Asthma is a chronic inflammatory disease characterized by T helper cell 2 (Th2) inflammation leading to

airway hyper-responsiveness (AHR). Evidence has indicated that Treg cells are involved in this disease49. Our data showed that GATA3, miR-125a-5p, FOXP3 and IL-6R are perturbed in the Treg

cells of asthma patients. Further studies on functional and tissue-specific Treg cell subsets of asthma patients would be meaningful to reveal the functional consequences of altering this

signal pathway. In conclusion, we have identified a novel signal pathway in which miR-125a-5p decreases the sensitivity of Treg cells toward IL-6-mediated conversion. This signal pathway

links two critical transcription factors in Treg cells with the function of one miRNA, which supports the notion that miRNAs are important regulators in Treg cells. Furthermore, miR-125a-5p

and IL-6R are perturbed in asthma patients, which provides basis for development of new therapeutic strategies against asthma. METHODS CELL CULTURE AND TRANSFECTION HEK293T cells were

cultured in DMEM containing 10% fetal bovine serum (FBS) and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Jurkat and SZ4 cell lines were

cultured in RPMI-1640 containing 10% FBS. Electroporation of Jurkat cells was performed with the NEPA21 apparatus (NEPAGENE, Japan). MIRNA MICROARRAY The microarray experiment was performed

using the Agilent-021827 Human miRNA Microarray. The microarray data analyzed for this publication has been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series

accession number; GSE64074 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=gjwremicrnkdjcl&acc=GSE64074) REAL-TIME POLYMERASE CHAIN REACTION ASSAYS Total RNA was extracted using

TRIzol reagent (Invitrogen). cDNA was synthesized using a reverse transcriptase kit (TaKaRa, Japan), followed by Real-Time-PCR analysis (SYBR Green; TaKaRa). The primers that were used are

as follows: ß-actin forward, 5′-GGACTTCGAGCAAGAGATGG-3′ and reverse, 5′-AGCACTGTGTTGGCGTACAG-3′; IL-6R forward, 5′-CCTGACGACAAAGGCTGTGCTCT-3′ and reverse, 5′-GCTGAACTTGCTCCCGACACTACTG-3′;

STAT3 forward, 5′-GGGGCTTTTGTCAGCGATGGAGTA-3′and reverse, 5′-ATTTGTTGACGGGTCTGAAGTTGAG-3′; GATA3 forward, 5′- CTCATTAAGCCCAAGCGAAG-3′ and reverse, 5′- TTTTTCGGTTTCTGGTCTGG-3′; FOXP3 forward,

5′-TCCCAGAGTTCCTCCACAAC-3′ and reverse, 5′-ATTGAGTGTCCGCTGCTTCT-3′. The expression level of miR-125a-5p was assayed with Hairpin-itTM MicroRNAs Quantitation PCR Kit (GenePharma, Shanghai,

China). Each sample was analyzed in triplicate and U6 RNA was used to normalize miRNA levels. WESTERN BLOT ANALYSIS Cells were washed with pre-chilled phosphate-buffered saline (PBS) and

lysed in radioimmune precipitation assay buffer. These cell lysates were separated on SDS-PAGE gels and transferred to PVDF membrane. All the primary antibodies were incubated overnight at 4

degrees Celsius, followed by incubation with HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibody and detected with ECL solution (Millipore). Antibodies against ß-actin

were purchased from Sigma; anti-STAT3 (79D7) was purchased from Cell Signaling. Anti-GATA3 (HG3-31) antibody was purchased from Santa Cruz Biotechnology. LUCIFERASE ACTIVITY ASSAY The human

3′ UTR regions of IL-6R or STAT3 were amplified by PCR and cloned into the EcoRI and XhoI sites or KpnI and XbaI sites of the pGL3-control vector (Promega). The promoter region of

miR-125a-5p was amplified by PCR and cloned into the KpnI and XhoI sites of the pGL3-basic vector (Promega). Nucleotide-substitution mutations were carried out using PCR-based methods. All

primers used in vector construction are listed in the supplemental tables. All constructs were verified by sequencing. For the luciferase assay, 293T cells were cultured in 12-well plates

and transfected with 100 ng luciferase reporter plasmid, 5 ng pRL-TK vector expressing the Renilla luciferase (Promega) and 25nM, 50nM or 100 nM of ago miR-125a-5p or anta miR-125a-5p or

miRNA negative control. For the promoter activity detection, 100 ng of luciferase reporter vector, 5 ng of pRL-TK vector and PIP-Flag-GATA3 or PIP-Flag-Blank vectors were co-transfected into

293T cells. After transfection for 48 hours, firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay (Promega). LENTIVIRAL CONSTRUCTS AND INFECTION

The shRNA lentiviral vectors pLKO.1 shGATA3-2 or pLKO.1 shCK were transfected into HEK 293T cells with the lentivirus packing vector Delta 8.9 and VSVG envelope glycoprotein. Viral

supernatants were harvested after 48 h. Primary human Treg cells were transduced with virus along with a secondary anti-CD3/CD28 stimulus (four cells to one bead). The following shRNA

sequence was used in this experiment: 5′-AGCCTAAACGCGATGGATATA-3′ (shGATA3-2). HUMAN T CELL CULTURE Naïve human CD4+ T cells (CD4+ CD25lowCD127highCD45RAhigh) and primary human CD4+

CD25highCD127low Treg cells from healthy donors were isolated by FACS on a BD FACS ARIA II sorter (BD Biosciences). Primary Treg cells were expanded using anti-CD3/CD28 dynabeads

(Invitrogen) in X-VIVO-15 medium (Lonza, Switzerland) supplemented with 10% human AB serum, 1% GlutaMax (GIBCO), 1% sodium pyruvate (GIBCO), 1% Pen/Strep (GIBCO) and 100 U/ml IL-2. Naïve T

cells were activated with anti-CD3/CD28 dynabeads in X-VIVO-15 medium and polarized into other T subsets under the following conditions: Th1: rhIL-12 (1 ng/ml) and anti-IL-4 (10 μg/ml)

antibody; Th2: rhIL-4 (20 ng/ml) and anti-IFNγ (10 μg/ml) antibody; Th17: rhTGF-β1 (2.5 ng/ml), rhIL-6 (50 ng/ml), rhIL-1β (10 ng/ml) and rhIL-23 (100 ng/ml) and iTreg: 10 ng/ml TGF-ß, 100 

ng/ml all-trans retinoic acid (atRA) and 100 units/ml rIL-2. SUPPRESSION ASSAY After culture for seven days, the Treg cells are restimulated with anti-CD3/CD28 dynabeads, 12.5 U/ml IL-2 and

with or without 20 ng/ml IL-6 for two days. To measure suppression function, CFSE-labeled PBMC cells were stimulated with anti-CD3/CD28 dynabeads. To the responder cells, Tregs stimulated

with or without IL-6 were added at different ratios and suppression of CFSE-labeled T cells was assessed as described50. ASTHMA PATIENTS AND TREG ISOLATION The human patients and healthy

control samples were from Ruijin Hospital (Shanghai, China). The study was approved by the Ruijin Hospital Ethics Committee, Shanghai Jiao Tong University School of Medicine (Permit number

2013-51) and was carried out in accordance with the approved guidelines. All participants provided written inform consent. The isolation of PBMC and Treg cells was performed as previously

described20. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Li, D. _et al._ MiR-125a-5p Decreases the Sensitivity of Treg cells Toward IL-6-Mediated Conversion by Inhibiting IL-6R and

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research is supported by the grants from National Basic Research Program of China (973 Program) 2014CB541803 and 2014CB541903, National Science Foundation of China 31200647, 81330072,

31370863, 31170825, 81270083, 31200646, 81271835, 81302532 and 31300711, Shanghai Postdoctoral Sustentation Fund 12R21417100, China Postdoctoral Science Foundation 2012M520946 and National

Science and Technology Major Project Grants 2012ZX10002007-003, 2013ZX10003009–002, SMCST 11ZR1404900 and 14JC1406100. We gratefully acknowledge the Knowledge Innovation Program of Shanghai

Institutes for Biological Sciences, Chinese Academy of Sciences 2012KIP204. AUTHOR INFORMATION Author notes * Li Dan and Kong Chao contributed equally to this work. AUTHORS AND AFFILIATIONS

* Key Laboratory of Molecular Virology & Immunology, Unit of Molecular Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,

Shanghai, 200031, China Dan Li, Chao Kong, Andy Tsun, Chen Chen & Bin Li * Shanghai Key Laboratory of Bio-energy Crops, College of Life Science, Shanghai University, Shanghai, 200444,

China Chao Kong * Department of Pulmonary Medicine, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200025, China Huihui Song & Guochao Shi * State Key

Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Wen Pan, Dai Dai & Nan Shen * Joint

Molecular Rheumatology Laboratory of the Institute of Health Sciences and Shanghai Renji Hospital, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai

Jiaotong University School of Medicine, Shanghai, 200025, China Wen Pan, Dai Dai & Nan Shen * Division of Rheumatology and the Center for Autoimmune Genomics and Etiology (CAGE),

Cincinnati Children’s Hospital Medical Center, Cincinnati, 45229, OH, USA Nan Shen * Department of Genetics, Yale University School of Medicine, New Haven, 06520, CT, USA Wen Pan Authors *

Dan Li View author publications You can also search for this author inPubMed Google Scholar * Chao Kong View author publications You can also search for this author inPubMed Google Scholar *

Andy Tsun View author publications You can also search for this author inPubMed Google Scholar * Chen Chen View author publications You can also search for this author inPubMed Google

Scholar * Huihui Song View author publications You can also search for this author inPubMed Google Scholar * Guochao Shi View author publications You can also search for this author inPubMed

 Google Scholar * Wen Pan View author publications You can also search for this author inPubMed Google Scholar * Dai Dai View author publications You can also search for this author inPubMed

 Google Scholar * Nan Shen View author publications You can also search for this author inPubMed Google Scholar * Bin Li View author publications You can also search for this author inPubMed

 Google Scholar CONTRIBUTIONS D.L. designed the research; B.L. supervised the research; D.L., C.K. and A.T. conducted the experiments; D.L. wrote the manuscript; A.T. revised the manuscript;

H.S. and G.S. provided clinical samples; C.C., W.P., D.D. and N.S. helped the experiments. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

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