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Journal of Autoimmunity 75 (2016) 96e104

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Journal of Autoimmunity

journal homepage: www.elsevier .com/locate/ jaut imm

Identification of the long noncoding RNA NEAT1 as a novelinflammatory regulator acting through MAPK pathway in humanlupus

Feifei Zhang a, b, 1, Lingling Wu a, 1, Jie Qian a, 1, Bo Qu a, Shiwei Xia a, Ting La b,Yanfang Wu a, Jianyang Ma a, Jing Zeng a, Qiang Guo a, Yong Cui d, Wanling Yang e,Jiaqi Huang f, Wei Zhu f, Yihong Yao f, Nan Shen a, b, c, *, Yuanjia Tang a, b, **

a Shanghai Institute of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, Chinab Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences (SIBS), ChineseAcademy of Sciences (CAS), Shanghai, Chinac The Center for Autoimmune Genomics and Etiology (CAGE), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USAd Institute of Dermatology and Department of Dermatology, No.1 Hospital, Anhui Medical University, Hefei, Chinae Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, Chinaf Cellular Biomedicine Group Inc., Shanghai, China

a r t i c l e i n f o

Article history:Received 23 January 2016Received in revised form18 July 2016Accepted 25 July 2016Available online 29 July 2016

Keywords:Long noncoding RNANEAT1TLR4Systemic lupus erythematosusCytokines

* Corresponding author. Shanghai Institute of RhShanghai Jiao Tong University School of Medicine, 1Shanghai, China.** Corresponding author. Shanghai Institute of RhShanghai Jiao Tong University School of Medicine, 1Shanghai, China.

E-mail addresses: nanshensibs@gmail.com (N. She(Y. Tang).

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jaut.2016.07.0120896-8411/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

Long noncoding RNAs (lncRNAs) have recently been identified to be tightly linked to diverse humandiseases. However, our knowledge of Systemic Lupus Erythematosus (SLE)-related lncRNAs remainslimited. In the present study we investigated the contribution of the lncRNA NEAT1 to the pathogenesisof SLE. Here, we found NEAT1 expression was abnormally increased in SLE patients and predominantlyexpressed in human monocytes. Additionally, NEAT1 expression was induced by LPS via p38 activation.Silencing NEAT1 significantly reduced the expression of a group of chemokines and cytokines, includingIL-6, CXCL10, etc., which were induced by LPS continuously and in late stages. Furthermore, it wasidentified the involvement of NEAT1 in TLR4-mediated inflammatory process was through affecting theactivation of the late MAPK signaling pathway. Importantly, there was a positive correlation betweenNEAT1 and clinical disease activity in SLE patients. In conclusion, the increased NEAT1 expression may bea potential contributor to the elevated production of a number of cytokines and chemokines in SLEpatients. Our findings suggest lncRNA contributes to the pathogenesis of lupus and provides potentiallynovel target for therapeutic intervention.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Systemic Lupus Erythematosus (SLE) is a chronic, multi-systemautoimmune disease [1,2]. Though lots of work has been done toelucidate the pathogenesis of SLE, the exact etiology of SLE is still

eumatology, Renji Hospital,45 Shan Dong Rd (Central),

eumatology, Renji Hospital,45 Shan Dong Rd (Central),

n), tangyuanjia028@163.com

unknown. Conventionally, SLE is characterized as a disease pri-marily caused by autoantibody production and immune complexdeposition. However, emerging evidence suggests the innate im-mune response is also crucial contributor to the pathogenesis of SLE[3e5].

Monocytes were a crucial component of innate immune. Acti-vation of monocytes results in the up-regulation of several surfacemolecules and the secretion of multiple monokines which partici-pate in various functions such as chemotaxis (eg, CCR2, MCP-1),adhesion (eg, ICAM-I), phagocytosis, antigen presentation and co-stimulation, modulation of lymphocyte effector functions, anddifferentiation to macrophages and dendritic cells (DCs) [6]. Evi-dences showed that monocytes severely altered in phenotype andlineage flexibility in SLE patients which indicated disturbances inthe monocytes initiating the auto-reactive cascade in SLE [7].

F. Zhang et al. / Journal of Autoimmunity 75 (2016) 96e104 97

Abnormal expression monocyte surface markers and cytokines(like IL-6)/chemokines (like CCL2, CCL5, CCL7, and CXCL10) in SLEwould be a key pathogenesis [8]. However, deep reason for thesealterations remained unknown.

Monocytes are equipped with an arsenal of conserved innatesensors designed to recognize pathogen-associated molecularpatterns (PAMPs) [9,10]. Activation of these receptors leads to theproduction of a wide spectrum of cytokines and chemokines.Through stimulating toll-like receptors (TLRs), especially TLR2 andTLR4, have been reported to contribute to the initiation andmaintenance of lupus disease. Lipopolysaccharide (LPS) is a Gram-negative cell wall component that can be recognized by TLR4. In SLEpatients, soluble CD14 (sCD14), which is released by monocytes inresponse to LPS, is increased in the blood [11]. The level of sCD14 ishighly correlated with disease activity parameters, suggesting theinvolvement of LPS in lupus development. In addition, experi-mental evidence in different animal models for SLE suggests a rolefor TLR4 in the development of murine lupus. Repeated injectionsof LPS into lupus-prone mice resulted in increased autoantibodyproduction and development of glomerulonephritis [12,13].Compared to C57BL/6lpr/lpr mice, TLR4-deficient C57BL/6lpr/lprmice develop a less severe disease, fewer immunological alter-ations, a diminished renal lesion, and significantly reduced anti-nuclear, anti-dsDNA, and anti-cardiolipin autoantibody titers [14].TLR4-deficient mice with pristane demonstrated a global decreasein cytokine production, less glomerular immunoglobulin andcomplement deposition [15]. In addition to binding exogenous li-gands derived from pathogens, accumulating evidence demon-strates that TLRs are also involved in SLE through interacting withendogenous ligands released from damaged tissues or dead cells,such as HMGB1, which interacts with TLR2 and TLR4 and correlateswith disease activity [16,17]. However, the detailed mechanism thathow dysregulated TLR4 signaling related to abnormal cytokinesand chemokines in monocytes from SLE was unclear, especially forthe aspect of long noncoding RNA, which was a newly emergingfield.

Long noncoding RNAs (lncRNAs) have been found to be preva-lent and important transcriptional outputs of the genome, andinvolved in diverse biological processes, such as epigenetic regu-lation and gene transcription [18e23]. Dysregulation of lncRNAswas found to have relevance to diverse human diseases rangingfrom neurological disorders to various cancers [24e27]. LncRNAswere also showed to be linked to autoimmune diseases, such asautoimmune thyroid disease (AITD) and rheumatoid arthritis (RA)[28,29]. Despite increasing evidence of lncRNAs playing roles indiverse biological processes and diseases, our knowledge of SLE-related lncRNAs remains limited.

Nuclear enriched abundant transcript 1 (NEAT1), a long non-coding RNA, is encoded on chromosome 11q13.1 and expressesconstitutively and widely in many tissues and cell types [30e32].Though NEAT1 is not detectable in human embryonic stem cells, itis induced upon differentiation [33]. Previous study has showedthat NEAT1 is essential for para-speckle structural integrity andformation through interacting with members of the DBHS(Drosophila Behavior Human Splicing) family proteins [34,35]. Sofar, NEAT1 has mainly been reported to control gene expressionthrough the nuclear retention of mRNA which contains invertedrepeats with adenosine-to-inosine editing at the 3’-end untrans-lated regions (UTR) [33,36].

Recently, emerging study has shown NEAT1 is closely linked toimmunity. At first, NEAT1 was known as VINC (virus induciblenoncoding RNA), which was initially detected in the brain of miceand further induced by virus infection [37]. Besides, researcherscharacterized a role contributed by NEAT1 to HIV-1 replication,which suggested that NEAT1 might play a pivotal role in response

to viral infection [38]. This evidence suggests NEAT1 plays animportant role in the innate immune response.

In this study, we observed a high expression of NEAT1 inmonocytes from patients with SLE. We found that NEAT1, an earlyLPS response gene, regulated the expression of inflammatory che-mokines and cytokines through the mitogen-activated protein ki-nase (MAPK) pathway. The findings presented here reveal therelevance of lncRNA NEAT1 in the biologic and clinical behavior ofan autoimmune disease-SLE.

2. Materials and methods

2.1. Patients and normal health controls

29 patients with SLE and 40 age-matched and sex-matchednormal controls were recruited for analyzing expression of theNEAT1 in PBMC. Another 10 patients with SLE and 10 age-matchedand sex-matched normal controls were recruited for isolatingPBMCs and then sorting monocytes by flow cytometry and testingexpression of NEAT1 in both PBMCs and monocytes. Healthy do-nors had no history of autoimmune diseases or treatment withimmunosuppressive agents. Patients with concurrent infectionwere excluded from the study. All SLE patients were recruited fromRenji Hospital and fulfilled the American College of Rheumatology(ACR) classification criteria for SLE. The Systemic Lupus Erythe-matosus Disease Activity Index (SLEDAI) score was determined foreach patient at the same time of their blood draw. Patients werecategorized as having active disease (scores>4) or inactive disease(scores�4) based on the SLEDAI results. The study was approved bythe Research Ethics Board of Renji Hospital, Shanghai Jiao TongUniversity School of Medicine. Informed consent was obtainedfrom all study participants. Additional clinical information on theSLE patients was given in supplementary materials.

2.2. Isolation of PBMCs, T cells, B cells and monocytes

Whole blood (10 ml) was collected in EDTA collection tubesfrom each subject, and PBMCs were isolated by density-gradientcentrifugation with Ficoll-Paque Premium (GE Healthcate), ac-cording to the instructions. For the subsets of PBMCs isolation, thefresh PBMCs were incubated for 15 min at 4 �C with flurescent-conjugated monoclonal antibodies: anti-CD3-PerCP-Cy5.5, anti-CD14-PE, anti-CD19-APC (all from BD Biosciences). Stained cellswere sorted on a BD FACSAria III (BD Biosciences). T cells wereidentified as CD3þ/CD19-. Monocytes were isolated if cells wereCD14þ/CD3-. B cells were collected if cells were CD19þ/CD3-. Thestained cells were sorted to >98% purity.

2.3. Cell culture and stimulation

The THP-1 cells were obtained from the Cell Bank, ShanghaiInstitutes for Biological Sciences. THP-1 cells were grown in RPMI1640 (Gibco, Life technology) with 10% fetal bovine serum (Gibco,Life technology) at 37 �C in a 5% CO2 atmosphere.

Two hours before stimulation, THP-1 cells were cultured in 24-well flat-bottomed plates at a concentration of 5 � 105 cells in500 mL of complete RPMI 1640 medium. Then cells were stimulatedfor 2 hwith the following stimuli: 100 ng/ml LPS (sigma),100 ng/mlPam3CSK4 (sigma), 25 mg/ml poly (I:C) (sigma). Or cells werestimulated with different concentration by LPS in indicated timepoints.

2.4. Transfection

Antisense oligonucleotides (ASOs) were designed by Sfold

F. Zhang et al. / Journal of Autoimmunity 75 (2016) 96e10498

(http://sfold.wadsworth.org) and synthesized by Sangon Biotech,Shanghai. The sequences of the ASO and scramblewere listed in thesupplementary materials. Two hours before transfection, THP-1 cells were cultured in 24-well flat-bottomed plates at a concen-tration of 105 cells in 500 mL of complete RPMI 1640 medium. ThenTHP-1 cells were transfected with the ASO or scramble at the finalconcentration of 200 nM using Lipofectamine RNAimax (Invi-trogen) according to the manufacturer's instructions for 12 h andthen cells were stimulated with LPS (100 ng/ml).

2.5. Real-time quantitative reverse transcription-PCR (RT-qPCR)

Total RNA was isolated with TRIzol reagent (Ambion). 200 ng oftotal RNA was reverse transcribed into Complementary DNA(cDNA) using a PrimeScript RT reagent kit with gDNA Eraser(Takara). Equal amount of cDNA was used for the subsequent qPCRperformed with the SYBR® Premix Ex Taq™ (Takara). Amplificationwas performed in an ABI vii7 Real Time PCR System (Applied Bio-systems). The expression of RPL13a was determined as the internalcontrol. Relative expression level was calculated using the 2�△Ct

method. Primers used were listed in the supplementary materials.

2.6. RNase protection assay (RPA)

For RPA, THP-1 cells were treated with LPS (100 ng/ml) for 2 hand then total RNA was isolated with TRIzol reagent. The RNaseprotection assay was performed as described before with the RPAIIIkit (Ambion) [35], according to the manufacturer's protocol. Briefly,10 mg of total RNAwas hybridized with a DIG-labeled antisense RNAprobe that was synthesized with DIG RNA Labeling Kit (Roche).RNase A/T1 digestion excluded unhybridized single-stranded RNAand probes. The protected RNA fragments were separated by 6%PAGE containing 7M urea. And then, separated RNAwas transferredand fixed tomembrane, and the following immunological detectionwas according to DIG Wash and Block Buffer Set (Roche). U12snRNA was hybridized as a loading control. The primers used toconstruct the plasmid DNA for labeling the RNA probes were listedin the supplementary materials.

2.7. Enzyme-linked immunosorbent assay (ELISA)

The protein levels of IL-6, CXCL10, CCL2 were measured at LPSstimulation (100 ng/ml) for 12 h. The protein levels of TNF-a weremeasured at LPS stimulation (100 ng/ml) for 2 h and 12 h. Theamount of IL-6, CCL2, TNF-a proteins secreted into the cell culturesupernatant was respectively quantified using commerciallyavailable Human ELISA DuoSet (R&D), according to the manufac-turer's protocol. The secretion of CXCL10 was detected by the hu-man CXCL10 ELISA kit (Westang, Shanghai) according to themanufacture's protocol.

2.8. Microarray analysis

THP-1 cells were transfected with ASO or scramble for 12 h andthen stimulated with 100 ng/ml LPS for 12 h. Total RNA wasextracted using TRIzol reagent and checked for a RIN number toinspect RNA integrity by an Agilent Bioanalyzer 2100. Qualifiedtotal RNA was further purified by RNeasy micro kit (QIAGEN) andRNase-Free DNase Set (QIAGEN). Total RNA was amplified, labeledand purified by using AffymetrixWTAmplication Kit and GeneChipWT Terminal Labeling Kit followed the manufacturer's instructionsto obtain biotin labeled cDNA. Array hybridization and wash wereperformed using GeneChip® Hybridization, Wash and Stain Kit(Affymetrix). Slides were scanned by GeneChip® Scanner 3000 andCommand Console Software 3.1 with default settings. Raw data

were normalized by Gene Spring Software 11.0.

2.9. Western blotting

THP-1 cells were seeded at 106/well in a 6-well plate andtransfected with ASO at the final concentration of 200 nM for 12 h.Then cells were stimulated with 100 ng/ml LPS for the indicatedtime, and the cells were lysed and the protein was extracted. Theprotein was subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis and blotted with antibodies. The antibodiesused were listed in the supplementary materials.

2.10. Statistic analysis

Data were analyzed using GraphPad Prism (version 5.01).Nonparametric Mann-Whitney test was used to compare geneexpression between SLE and normal controls. The Student's un-paired t-test was used to compare the relative mRNA and proteinlevels of LPS stimulated genes with or without silencing NEAT1 inTHP-1 cells. Nonparametric correlation (spearman) was done toanalyze associations between NEAT1 expression and SLEDAI scoreor expression of NEAT1-regulated genes in SLE. P values (2-tailed)less than 0.05 were considered to be statistically significant.

3. Results

3.1. NEAT1 expression is increased in SLE patients

To characterize NEAT1 function in SLE, first we measured theexpression of NEAT1 in the peripheral blood mononuclear cells(PBMCs) obtained from 39 SLE patients and 50 normal controls.Since NEAT1 had two isoforms, a smaller 3.7 kb (NEAT1-V1) and alarger 23 kb isoform (NEAT1-V2) [30]. So we used two pairs ofprimers (Fig. 1A) to detect these two transcripts. As shown inFig. 1B, the total NEAT1 expressionwas abnormally increased in SLEpatients as compared with normal controls. And we also found anincrease of NEAT1-V2 expression in the SLE patients (Fig. S1A).Besides, medications do not affect the expression of NEAT1 andNEAT1 was intrinsically overexpressed in lupus patients (Fig. S1Band C). Through the analysis of the relative expression of NEAT1,it was showed that NEAT1-V2 was of small percentage of NEAT1and NEAT1-V1 was the main transcript which agreed with theformer studies about NEAT1 expression in different tissues [32,39].

Considering the differential expression of NEAT1 in the subsetsof PBMCs, we examined the expression of NEAT1 in the mainsubsets of PBMC (T cells, monocytes and B cells) from 10 healthydonors. As shown in Fig. 1C, monocytes expressed especially highlevel of NEAT1. In addition, considering the increased percentage ofmonocytes in PBMC of SLE patients [40], to exclude that the dif-ference in NEAT1 expression in PBMC is caused merely by thisfactor, we tested NEAT1 expression in monocytes from SLE patientsand healthy controls. And we found NEAT1 was also significantlyup-regulated in monocytes of SLE patients as compared withnormal controls (Fig. 1D). As secretion inflammatory cytokines inresponse to innate immunity ligands is one of the prominent fea-tures of monocytes. Based on above evidence, we speculated thatNEAT1 might contribute to the pathogenesis of SLE through itsunknown function in monocytes.

3.2. NEAT1 is an early-response gene induced by different TLRligands including LPS

NEAT1 was reported to be induced by poly (I:C), a double-stranded RNA mimicking immunostimulant that simulated viralinfections in Hela TO cells [41]. As emerging evidence reported that

Fig. 1. NEAT1 expression is increased in SLE patients. A, Graphical representation of human NEAT1 transcripts, qPCR primers, ASO and siRNA mapping to the NEAT1 transcripts. B,Expression of NEAT1 in PBMCs of SLE patients (n ¼ 39) and normal controls (NC) (n ¼ 50), as determined by qPCR analysis. C, Expression of NEAT1 in T cells, monocytes and B cellsfrom 10 healthy donors. D, NEAT1 expressionwas up-regulated in monocytes of SLE patients (n ¼ 10) compared with normal controls (n ¼ 10). The relative expression of NEAT1 wasdetermined by qPCR. Bars showed the mean ± SEM and P values were determined by nonparametric Mann-Whitney test.

F. Zhang et al. / Journal of Autoimmunity 75 (2016) 96e104 99

stimulating TLRs contributed to the initiation and maintenance oflupus disease [42], we wondered that whether TLR signaling acti-vation in SLE patients related to increased expression of NEAT1 inmonocytes from SLE patients. Considering the expression level ofTLRs in circulating monocytes [43], we stimulated the THP-1 hu-man monocytic cell line which has been used extensively to studythe innate immune response with various innate immunity ligands[44,45], and found that the expression of NEAT1 was significantlyincreased after treatment with LPS or pam3cks4 for two hours(Fig. 2A). And stimulation of TLR3 had no apparent effect on NEAT1expression in THP-1 cells (Fig. 2A) which may be caused by lackingTLR3 expression in THP-1 cells [46]. We also did these experimentsin human primary monocytes freshly isolated from PBMCs andfound that different agonistic ligands of TLRs could induce theexpression of NEAT1 including TLR3 ligands (Fig. S2A). Further-more, as shown by qPCR, we found that induction of NEAT1 afterLPS challenge was in a dose-dependent manner and peaked at 2 h,suggesting that NEAT1 might be a TLR early-response gene in bothTHP-1 cell lines (Fig. 2B) and human primary monocytes (Fig. S2B).Because the short isoform of NEAT1 (NEAT1-V1) has a total overlapwith the 50 end of the longer one (NEAT1-V2), it's difficult todistinguish them by qPCR. To identify the induction of the twodifferent isoforms respectively, we used RNase protection assay(RPA) to separate them as previously described [28]. The strategyand probe position used were shown in Fig. 2C. RPA analysisshowed that the expression of both NEAT1-V1 and NEAT1-V2 wasup-regulated after 2 h of LPS treatment (Fig. 2D).

It has been demonstrated that TLR4 stimulation can lead to theprimary response through activating the NF-kB and MAPKpathway. To identify the pathways that regulated NEAT1 expressionin THP-1 cells, we pretreated THP-1 cells with a NF-kB inhibitor(PDTC), an ERK inhibitor (U0126), a JNK inhibitor (SP600125) or ap38 inhibitor (SB203580) and then exposed them to LPS for 2 h. The

result showed only pretreatment of p38 inhibitor reduced LPS-triggered NEAT1 induction (Fig. S3A). We further found that theLPS-triggered NEAT1 induction was reduced in p38-silenced cellscompared with control cells (Fig. S3B). The results suggest the in-duction of NEAT1 by LPS is mainly dependent on the TLR4-p38pathway.

3.3. NEAT1 regulates IL-6 production after LPS stimulation

Considering NEAT1 was up-regulated in response to LPS, wewondered whether or not NEAT1 would be involved in regulationof LPS-triggered induction of numerous inflammatory factors,including IL-6 and TNF-a [47]. To reveal the role of NEAT1 in LPS-triggered inflammatory cytokines production, we silenced NEAT1with target-specific ASO in THP-1 cells and stimulated the cells byLPS (Fig. 3A). IL-6 and TNF-a mRNA levels were then measured byqPCR, and the protein levels in the supernatants were measured byELISA. As shown in Fig. 3B, silencing NEAT1 significantly reducedLPS-induced expression of IL-6. However, silencing NEAT1 had noapparent effect on the expression of LPS-induced TNF-a (Fig. 3C).Besides, we silenced NEAT1 with siRNA as described before [34],and found NEAT1 selectively regulated IL-6, instead of TNF-aexpression in response to LPS (Fig. S4), consistent with the resultsshown in Fig. 3.

3.4. Identification of NEAT1-regulated genes

To overview the landscape of genes whichmight be regulated byNEAT1, we analyzed the expression profiles of the LPS stimulatedTHP-1 cells with or without silencing NEAT1. Microarray analysisrevealed 183 NEAT1-regulated protein coding genes (Fig. 4A, B). GOenrichment and KEGG pathways analysis revealed significantinvolvement of biological processes such as chemokine activity and

Fig. 2. NEAT1 expression is induced by LPS. A, Analysis of NEAT1 expression in response to various innate immunity ligands (Pam3CSK4, 100 ng/ml; PolyI:C, 25 mg/ml; LPS, 100 ng/ml) for 2 h in THP-1 cells by qPCR. Bars showed the mean ± SEM of three independent experiments and P values were analyzed with two-tailed unpaired t-test. **P < 0.01. B,Kinetics of up-regulation of NEAT1 in response to different concentrations of LPS in THP-1 cells for the indicated times. NEAT1 expression was analyzed by qPCR and bars showedthe mean ± SEM from three independent experiments. C, Graphical representation of RPA probe mapping to the NEAT1 transcripts. D, RPA analysis was performed to distinguish theexpression of NEAT1-V1 and NEAT1-V2 in response to LPS. THP-1 cells were treated with LPS for 2 h. NEAT1 probe was hybridized with total RNA. U12 snRNA was hybridized as aloading control.

Fig. 3. NEAT1 regulates the expression of IL-6 A, The efficiency of silencing NEAT1 by ASO was analyzed by qPCR. THP-1 cells were transfected with ASO or scramble for 12 h andthen stimulated with LPS (100 ng/ml) for the indicated time. B, The IL-6 mRNA expression (left) in THP-1 cells with NEAT1 ASO or scramble was determined by qPCR. IL-6 in thesupernatants (right) of the LPS-stimulated THP-1 cells for 12 h was measured by ELISA. C, TNF-a mRNA expression (left) and protein levels (right) with silencing NEAT1 after LPSstimulation were measured by qPCR and ELISA respectively. Bars showed the mean ± SEM from three independent experiments and P values were analyzed with two-tailedunpaired t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

F. Zhang et al. / Journal of Autoimmunity 75 (2016) 96e104100

cytokine activity reflected by these genes (Fig. 4C, D).Then we verified the induction of CXCL10 and CCL2 in THP-

1 cells, two selected genes regulated by NEAT1 from themicroarray,

by qPCR and ELISA (Fig. S5A, S5B). At the same time, we noted thatCXCL10 and CCL2were induced continuously and in a late-responsemanner and the induction rose to a higher level 12 h after LPS

Fig. 4. Identification of NEAT1-regulated genes. A, Schematic sketch of microarray analysis of gene expressions. B, Heat map of microarray analysis of gene expression in the controlcells and NEAT1 knockdown cells with no stimulation or LPS stimulation. C, GO enrichment of the variable genes. D, KEGG pathways analysis of the microarray data.

F. Zhang et al. / Journal of Autoimmunity 75 (2016) 96e104 101

stimulation. To determine the kinetic expression of the NEAT1-regulated genes after LPS stimulation, we measured a number ofadditional NEAT1-regulated genes such as CXCL11, CCL8 and ISG20,and found that all of these genes were induced slowly andcontinuously until 12 h (Fig. S5C), in accordancewith the kinetics ofIL6, CCL2 and CXCL10 we mentioned above. However, the expres-sion of genes which peaked at 2 h and fell to a relatively low level at12 h after LPS stimulation were not regulated by NEAT1, such asCXCL1, IL-12b and IL-8 (Fig. S5D). These results suggest NEAT1selectively regulates expression of a set of LPS-induced genes whichare induced continuously and at late stages. We also knocked downNEAT1 in primary monocytes by using electro-transfectionmethods (Fig. S6A), and found that down-regulated NEAT1 wouldsuppress IL-6, CXCL10, CCL8 rather than CXCL1 (Fig. S6B), partiallyconsistent with the results shown in THP-1 cells.

3.5. NEAT1 affects the activation of the late MAPK pathways

Since NEAT1 could mediate the set of LPS continuously-inducedand late-response genes, we wondered whether NEAT1 wouldaffect the TLR4-triggered secondary response which needed thenewly synthetic proteins. So we added cycloheximide (CHX) toblock protein translation, and found that without new proteinsynthesis silencing NEAT1 still reduced the expression of IL-6, CCL2and CXCL10 (Fig. S7). The result indicates that the function ofNEAT1 is not through the secondary response, but mainly involvedin the primary response.

To figure out which pathway of the TLR4-triggered primaryresponse NEAT1 was involved in, we detected the phosphorylationof the key members of the pathways. And we found that TLR4-triggered phosphorylation of JNK1/2, ERK1/2, p38 and p65 werenot significantly affected by silencing NEAT1 in the early 90minafter LPS treatment (Fig. 5A, Fig. S8). However, whenwe treated theTHP-1 cells for a longer time, the phosphorylation of members ofMAPK pathways, in especial JNK1/2 and ERK1/2, was significantlyreduced with silencing NEAT1 compared with control cells (Fig. 5B,

Fig. S8). These results suggest NEAT1 selectively regulates TLR4continuously-induced and late-response genes mainly throughaffecting the activation of the late MAPK pathways, especially theactivation of JNK and ERK.

3.6. A positive correlation between NEAT1 and NEAT1-regulatedgenes or disease activity in SLE patients

Since NEAT1 could affect the late MAPK pathway activation andconsequently regulate a set of LPS-induced cytokines and chemo-kines which were dysregulated in patients with SLE, we wonderedwhether there was any relationship between NEAT1 and NEAT1-regulated genes in SLE patients. As shown in Fig. 6A and B, a pos-itive correlation was observed between NEAT1 and IL-6 or CXCL10in SLE patients. Furthermore, to determine whether there was anyrelationship between NEAT1 and disease activity, we compared theexpression of NEAT1 in PBMCs from inactive SLE patients with thatof the active SLE patients. As shown in Fig. 6C, the expression ofNEAT1 was higher in active SLE patients than that of inactive pa-tients. And there was a positive correlation between NEAT1expression and SLEDAI score in patients with SLE (Fig. 6D). Theseresults indicate that in SLE patients, the overexpressed NEAT1 levelcorrelates positively with the disease activity and NEAT1-regulatedcytokines and chemokines.

4. Discussion

The innate immune system is the host's immediate line of de-fense against both endogenous and exogenous molecules.Increasing evidence shows the innate immune response contrib-utes to the development and regulation of selected autoimmunediseases, including SLE [3,4,48e51]. Monocytes were one of themajor components of innate immune system. Evidence showedthat monocytes severely altered in phenotype and lineage flexi-bility in SLE patients [7]. Abnormal expression of the monocytesurface markers and cytokines (like IL-6)/chemokines (like CCL2,

Fig. 5. NEAT1 affects the activation of the late MAPK pathways, in especial the phosphorylation of JNK and ERK. A, Western blotting of the phosphorylation of JNK1/2, ERK1/2, p38or p65 after LPS stimulation for the first 90min. THP-1 cells were transfected with ASO or scramble for 12 h and then stimulated with LPS (100 ng/ml) for the indicated times. B,Silence of NEAT1 repressed the phosphorylation of JNK1/2, and ERK1/2. Western blotting analysis of the activation of the pathway for the 120mine300min after LPS stimulation(100 ng/ml). Quantification of band intensities analysis was shown in Fig. S8.

Fig. 6. A positive correlation between NEAT1 expression and NEAT1-regulated genes or disease activity in SLE patients. Nonparametric correlation analysis was performed to assessthe correlation between the expression of NEAT1 and NEAT1-regulated genes IL-6 (A) CXCL10 (B) in PBMCs of patients with SLE (n ¼ 39). C, NEAT1 expressions in PBMCs of 13patients with inactive SLE, 26 patients with active SLE and 50 normal controls were analyzed by qPCR. P values were determined by nonparametric Mann-Whitney test. D,Nonparametric correlation (spearman) was performed to assess the correlation between NEAT1 and SLEDAI score in patients with SLE (n ¼ 39).

F. Zhang et al. / Journal of Autoimmunity 75 (2016) 96e104102

CCL5, CCL7, and CXCL10) in SLE would be a key pathogenesis [8].Accumulating researches indicate that lncRNAs play importantroles in innate immune system [52,53]. In the present study, wefound nuclear lncRNA NEAT1 was aberrantly up-regulated inmonocytes from lupus patients. To our knowledge, this is the firstpiece of evidence showing the lncRNA participates in the patho-genesis of SLE through dysregulating proinflammatory chemokinesand cytokines by affecting TLR4-mediated inflammatory pathway

in monocytes of SLE patients. Besides, considering that NEAT1 wasconserved between human and mice and it was up-regulated insome lupus mice model (data not shown here), more work need tobe done to explore the specific function in vivo study.

By exploring the function of NEAT1, we found NEAT1 selectivelyregulated a subset of genes which were involved in immune dys-regulation and local inflammation leading to target tissue damagein SLE. Among of these NEAT1-regulated genes, their functions

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were with enrichment as chemokine activity. Chemokines playpivotal roles in the recruitment of inflammatory cells into sites ofinflammation. In SLE, there has been growing evidence suggestingthat infiltration of T lymphocytes into the sites of the kidney plays acritical role in organ damage. Lupus nephritis is characterized byglomerular T helper 1 (Th1) cells accumulation and it is notable thatthe dominantly expressed CCR2, CCR5 and CXCR3 in Th1 cells arereceptors of the NEAT1-regulated chemokines (CCL2, CCL3, CCL4,CCL8, CXCL9, CXCL10, CXCL11) [54e57]. The evidence provides usclues that NEAT1 may facilitate the pathogenesis of SLE throughpromoting the crucial chemokines involved in the immune dysre-gulation in SLE. Besides, we have also noticed a large number of theNEAT1-regulated genes are a panel of type I interferon inducedgenes including IFI27, OAS1, CXCL9, IFI35, CXCL10, CXCL11 and soon. This may indicate NEAT1 plays a role in the type I interferonpathway which is key contributor in the pathogenesis of SLE.

In addition to innate immune system, we also found that NEAT1expression increased in total CD3þ T cells from SLE patientscompared to normal control (data unpublished). There was aprofiling research to compare gene expression in CD4þ Tcells, CD8þ

T cells and double negative (DN) T cells [58], and we retrievedNEAT1 probe in this array data and found that NEAT1 was widelyexpressed in those three T cell subpopulation. In a previous study,DN Tcells were shown to produce the inflammatory cytokines IL-17and IFN-gamma, and contribute to the pathogenesis of kidneydamage in patients with SLE [59]. Whether NEAT1 functions in Tcells, especially DN T cells, would be an interesting and valuabletopic, which need to be further explored.

In this study, we found that the set of genes regulated by NEAT1were induced by LPS continuously and at late stages. Meanwhile,the effect of NEAT1 on LPS-induced genes was not through thesecond response which needed the newly synthetic proteins.However, the early response genes like CXCL1 and IL-8 were notaffected with silencing NEAT1. More recently, a report showedNEAT1 regulated the expression of IL-8 depending on relocating theSFPQ binding to the IL-8 promoter in Hela TO cells [41]. Unfortu-nately, in THP-1 cells, IL-8 was an early response gene in responseto LPS and not apparently affected with silencing NEAT1, whichdiffered from the aforementioned observation in Hela TO cells. Wesupposed that NEAT1 might function in different manners in spe-cific cells which needed to be further explored. To figure out howNEAT1 selectively regulated LPS-induced genes, we detected theLPS-triggered phosphorylation of the key members of the pathway.The intracellular signaling pathways of LPS-induced inflammationare known as NF-kB pathway and the MAPK pathways (ERK, JNKand p38). To gain insight of the effect of NEAT1 on LPS-inducedsignaling, we investigated whether MAPKs and NF-kB pathwayswere regulated by NEAT1 and found that silencing NEAT1decreased LPS induced-MAPK phosphorylation, in especial thephosphorylation of ERK and JNK at the late stage of LPS stimulation.Though the thoroughly molecular mechanism is currently un-known, we speculate that NEAT1, early induced by LPS through p38signaling pathway, enhances the late phase phosphorylation of theMAPK signaling pathways, especially the phosphorylation of JNKand ERK, resulting in the overproduction of the late andcontinuously-induced cytokines and chemokines (Fig. S9).Furthermore, a previous study demonstrated that activity of bothERK and JNK was increased in SLE patients and reflected diseaseactivity [60]. We infer that up-regulated expression of NEAT1 mayresult in the abnormal activation of MAPK signaling pathways inSLE.

In our study, NEAT1 was a positive regulator in TLR4 signaling.As increased NEAT1 expression in monocytes and increasedmonocytes percentage of PBMCs from SLE patients compared tonormal controls, we would speculated that more monocytes

expressed highly NEAT1 in PBMC from SLE patients, more cytokinesand chemokines were produced by them, which was positivelyrelative to disease activity and disease pathogenesis. Thus, NEAT1would be a potential biomarker of disease diagnosis and a potentialhint of disease activity.

5. Conclusions

Our findings suggest the lncRNA NEAT1, as an early-responsegene, can mediate the expression of a subset of genes induced byLPS. In SLE patients, NEAT1 may participate in the immune dysre-gulation through acting as a newly identified proinflammatoryfactor to facilitate the overproduction of cytokines and chemokinesin a positive feedback manner. This research also brings us to theknowledge that lncRNAs may contribute to a new layer of molec-ular regulation of autoimmune diseases. What's more, under-standing the functions of the lncRNAs involved in the autoimmunedisease may be of clinical significance of identifying the lncRNAs asbiomarkers of disease activity and potential targets of therapy.

Author contributions

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authorsapproved the final version to be published. Study conception anddesign: Shen, Tang. Acquisition of data: Zhang, Qu, Xia, La, Wu,Qian, Zeng, Ma. Analysis and interpretation of data: Zhang, Wu,Qian, Guo, Cui, Yang, Zhu, Huang, Yao, Shen, Tang.

Acknowledgement

We thank Prof. Xiao Hui, Xu Chenqi, Zhang Xiaoren for theircritical reading of the manuscript and helpful comments. This workwas partially supported by the National Basic Research Program ofChina (973 program) (2014CB541901, 2014CB541902); NationalNatural Science Foundation of China (No. 81571576, No. 81230072,No. 31370880, No. 81421001); the Grants from the State Key Lab-oratory of Oncogenes and Related Genes (No. 91-14-05) and the KeyResearch Program of the Chinese Academy of Sciences (KJZD-EW-L01-3).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jaut.2016.07.012.

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