The long noncoding RNA MALAT1 regulates thelipopolysaccharide-induced inflammatory responsethrough its interaction with NF-jBGui Zhao1,*, Zhenyi Su1,*, Dan Song1, Yimin Mao1,2 and Xiaohua Mao1

1 Department of Biochemistry, School of Medicine & Key Laboratory of Ministry of Education for Developmental Genes and Human

Diseases, Southeast University, Nanjing, China

2 School of Life Science and Technology, China Pharmaceutical University, Nanjing, China

Correspondence

X. Mao, Department of Biochemistry,

School of Basic Medical Sciences,

Southeast University at Dingjiaqiao Campus,

Nanjing, Jiangsu 210009, China

Fax: +86 25 83324887

Tel: +86 25 83272474

E-mail: xmao@seu.edu.cn

*These authors contributed equally to this

work.

(Received 6 May 2016, revised 28 June

2016, accepted 11 July 2016, available

online 4 August 2016)

doi:10.1002/1873-3468.12315

Edited by Kazuhiro Iwai

MALAT1 is a conserved long noncoding RNA whose expression correlates

with many human cancers. However, its significance in immunity remains lar-

gely unknown. Here, we observe that MALAT1 is upregulated in lipopolysac-

charide (LPS)-activated macrophages. Knockdown of MALAT1 increases

LPS-induced expression of TNFa and IL-6. Mechanistically, MALAT1 was

found to interact with NF-jB in the nucleus, thus inhibiting its DNA binding

activity and consequently decreasing the production of inflammatory cyto-

kines. Additionally, abnormal expression of MALAT1 was found to be NF-

jB-dependent. These findings suggest that MALAT1 may function as an

autonegative feedback regulator of NF-jB to help fine-tune innate immune

responses.

Keywords: inflammation; innate immunity; Macrophage; MALAT1;

NF-jB transcription factor

The innate immune system is the major contributor to

inflammation induced by microbial infection. It recog-

nizes pathogens through the detection of structures

conserved among microbial species, which are called

pathogen-associated molecular patterns (PAMPs).

These molecular structures of microorganisms are

sensed by pattern recognition receptors (PRRs), of

which the Toll-like receptor (TLR) family is best char-

acterized [1]. Antigen-presenting cells express TLRs

and are especially sensitive to the products of microbes

bearing PAMPs such as lipopolysaccharide (LPS),

lipoproteins, and nucleic acids. The intracellular signal-

ing cascades triggered by these TLRs result in the

activation of transcription factors NF-jB, AP-1,

IRF3, and/or IRF7, which consequentially induce pro-

inflammatory mediators [e.g. tumor necrosis factor

alpha (TNFa), interleukin (IL)-1, and IL-6] that coor-

dinate the elimination of pathogens and infected cells.

However, an excessive inflammatory response impairs

resolution and can lead to chronic inflammation and

subsequent tissue damage. Thus, TLR signaling must

be tightly regulated to avoid detrimental and inappro-

priate inflammatory responses [1,2].

So far several mechanisms controlling TLR activity

have been uncovered. Some signaling pathways, such

as Notch [3], integrin CD11b [4], immunoreceptor

Abbreviations

ChIP, chromatin immunoprecipitation; DCs, dendritic cells; ECL, enhanced chemiluminescent; HRP, horseradish peroxidase; IL, interleukin;

lncRNAs, long noncoding RNAs; LPS, lipopolysaccharide; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition recep-

tors; qPCR, quantitative PCR; RIP, RNA immunoprecipitation; TLR, Toll-like receptor; TNFa, tumor necrosis factor alpha.

2884 FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

tyrosine-based activation-associated receptors [5], the

kinases MSK1 and MSK2 [6], cross-talk with TLR sig-

naling pathways, resulting in fine tuning of TLR-trig-

gered innate inflammatory responses. In addition, a

number of negative regulators have been identified to

regulate TLR signaling at multiple levels, ranging from

extracellular decoy receptors to intracellular inhibitors,

nuclear receptors, epigenetic regulators, membrane-

bound suppressors, degradation of TLRs, and TLR-

induced apoptosis [2,7–9]. Notably, many regulators

targeting various stages of the TLR signaling pathways

are induced by TLR ligands to maintain immune

homeostasis in a negative feedback manner [6,10–16].In addition, microRNA have been recently reported to

constitute an additional layer of the negative feedback

mechanism operating in the TLR pathway [17,18].

This effective self-control mechanism contributes

significantly to the overall balance between pro- and

anti-inflammatory responses. Of all the TLRs, TLR4

seems to be most heavily regulated probably due to

the extreme toxic and potentially lethal effects of

TLR4 signaling.

There is accumulating evidence that long noncoding

RNA (lncRNA) are important regulators of funda-

mental biological processes; however, their potential

importance in immune responses, particularly in innate

immune response, is only now emerging [19,20]. At

present, it appears that the action of most innate

immune-related lncRNAs is mediated through binding

to proteins including signaling molecules, transcription

factors, heterogeneous nuclear ribonucleoproteins, and

components of chromatin-modifying complexes [20].

Notably, PACER and NKILA, two lncRNAs that are

induced upon TLR ligand stimulation, were found to

regulate gene expression by interacting with key com-

ponents of NF-jB signaling [21,22], a pathway that

plays its most important and evolutionarily conserved

role in the immune system.

In this study we report a critical role of the highly

conserved lncRNA MALAT1 (metastasis-associated

lung adenocarcinoma transcript 1) in regulating the

human macrophage response to an innate stimulus.

Using the human THP1 monocyte cell line and a

PMA-driven monocyte-macrophage differentiation sys-

tem, we show that MALAT1 is upregulated in

response to LPS, a TLR4 ligand, in a NF-jB-depen-dent manner. MALAT1 interacts with NF-jB subunits

p65 (RelA) and p50 to inhibit NF-jB DNA binding

activity and the production of proinflammatory cytoki-

nes TNFa and IL-6. These findings suggest that

MALAT1 may function as a novel negative feedback

regulator of TLR signaling to help fine-tune inflamma-

tory reactions and innate immune responses.

Materials and methods

Cell culture

THP1 cells were cultured in RPMI-1640 (Hyclone) con-

taining 10% (v/v) FBS and differentiated into macro-

phages by treatment with 40 ng�mL�1 PMA for 24 h.

Then, the medium was replaced with fresh medium

without PMA. RAW264.7 macrophages were cultured in

Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone)

containing 10% FBS. To induce inflammatory response,

THP1 macrophages, RAW264.7 cells, or murine dendritic

cells (DCs) were treated with 100 ng�mL�1 LPS (Escheri-

chia coli 055:B5, Sigma-Aldrich, St. Louis, MO, USA)

unless indicated otherwise.

Dendritic cell preparation

Bone marrow-derived DCs were isolated from C57BL/6

mice by flushing femurs with RPMI 1640, washed and cul-

tured in 6-well plates at 4 9 106 cells per well in 2 mL of

complete medium (RPMI 1640 supplemented with 2 mM L-

glutamine, 100 U�mL�1 penicillin, 100 lg�mL�1 strepto-

mycin, 50 lM 2-ME, and 10% FBS) in the presence of

recombinant mouse granulocyte macrophage colony-stimu-

lating factor (10 ng�mL�1; Canspec, Shanghai, China) and

recombinant mouse IL-4 (10 ng�mL�1; Canspec). On day 2

and day 4, half of the medium was replaced with fresh

complete medium containing the cytokines. On day 6, the

immature DCs were washed and plated in 12-well plates at

8 9 105 cells per well. After 24 h, the cells were treated

with LPS for 6 h before being harvested for RNA extrac-

tion. Animal experiments were performed in accordance

with the guideline of the Committee on Animals of South-

east University, China.

siRNAs and primers

All siRNAs and PCR primers are listed in Table S1. Scram-

ble siRNA or siRNA targeting human MALAT1 (siMA-

LAT1) were obtained from Genepharma (Shanghai, China).

Unless indicated otherwise, siMALAT1-1 was chosen to

knockdown MALAT1 expression. DNA oligonucleotide pri-

mers were obtained from GenScript (Nanjing, China).

siRNA transfection

siRNA transfection was performed using X-tremeGENE

siRNA Transfection Reagent (Roche, Mannheim, Ger-

many) according to the manufacturer’s instructions. In

brief, PMA-differentiated THP1 cells were plated at

6 9 105 cells per well in 12-well plates. 1 lg of siRNA was

transfected by 5 lL of X-tremeGENE siRNA Transfection

Reagent for each well.

2885FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

G. Zhao et al. MALAT1 regulates the innate immune response

RNA extraction and real-time quantitative PCR

Total RNA was extracted using RNAiso Plus (Takara,

Dalian, China) according to the manufacturer’s instruc-

tions, and was reversely transcribed into first-strand cDNA

using PrimeScript RT reagent Kit with gDNA Eraser

(Takara). Real-time PCR was performed with Roche

SYBR Green I Master Mix. Expression of mRNA was nor-

malized to the expression of human b-actin or mouse

GAPDH (mGAPDH). Primer pairs for quantitative PCR

(qPCR) are listed in Table S1.

ELISA for secreted TNFa, IL-6 and IL-1b

THP1 macrophages were transfected with indicated siRNA

for 48 h and then treated with 100 ng�mL�1 LPS for 10 h.

The culture supernatants were collected and the level of

each cytokine was measured by ELISA (R&D Systems)

according to the manufacturer’s instructions.

Western blot

THP1 macrophages were transfected with either scramble

siRNA or siRNA targeting human MALAT1. At 48 h after

transfection, cells were treated with LPS for 10 h. Whole

cell lysates were subjected to western blotting using anti-p65

(sc-372, Santa Cruz Biotechnology, San Diego, CA, USA),

anti-p50 (sc-1190x, Santa Cruz Biotechnology) or anti-

b-actin (20536-1-AP, Proteintech, Chicago, IL, USA)

antibodies. Signals were visualized using a horseradish

peroxidase (HRP)-labeled secondary antibody (sc-2313,

Santa Cruz Biotechnology) and the enhanced chemilumines-

cent (ECL) detection systems (Pierce, Rockford, IL, USA).

Dual-luciferase assay

THP1 macrophages at 70% confluence were cotransfected

with indicated siRNA, NF-jB luciferase plasmid and

Renilla luciferase plasmid in a 24-well plate using Lipofec-

tamine2000 (Invitrogen, Carlsbad, CA, USA) according to

the manufacturer’s instructions. Each condition was per-

formed in triplicate. After transfection, the serum free

transfection medium was replaced with serum-containing

growth medium for 24 h. After 24 h, cells were left

untreated or treated with 100 ng�mL�1 LPS for 10 h. Cells

were then lysed and analyzed using Dual-Luciferase Repor-

ter Assay System (Promega, Madison, WI, USA). Lucifer-

ase values were normalized to Renilla to control for

transfection efficiency.

Generation of MALAT1 promoter reporter

constructs

Reporter genes containing the truncated fragments (�828/

+13, �349/+13, �324/+13) of the MALAT1 promoter

region were prepared by PCR amplification of human

genomic DNA of THP1 cells. After confirmation by

sequencing, the MALAT1 promoter fragments were direc-

tionally cloned into the pGL4.17-basic firefly luciferase vec-

tor (Promega). To prepare NF-jB-binding site mutation,

two nucleotides (�325/�326) in the putative NF-jB-bind-ing sequence were mutated from CC to GG by the method

of two-step PCR, using primers jB-mut F and R

(Table S1) and the wild-type MALAT1 promoter as a tem-

plate. THP1 macrophages were transiently transfected with

different MALAT1 promoter reporter constructs and

Renilla luciferase plasmid (Promega) at 50:1 using Lipofec-

tamine 2000 (Invitrogen). Each condition was performed in

triplicate. 24 h after transfection, cells were treated with

LPS for 6 h. Cells were then harvested and assayed using

Dual-Luciferase Reporter Assay System (Promega).

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed using

the Magna ChIP G Chromatin Immunoprecipitation Kit

(Catalog # 17-611, Millipore, Billerica, MA, USA) according

to the manufacturer’s instructions. Briefly, cross-linked chro-

matin was sonicated into DNA fragments in the range of

200–1000 bp. The chromatin was immunoprecipitated using

normal rabbit IgG (sc-2027, Santa Cruz Biotechnology) or

anti-p65 antibody (sc-372, Santa Cruz Biotechnology). qPCR

was performed with Roche SYBR Green I Master Mix using

primers listed in Table S1.

Nuclear and cytoplasmic RNA fractionation

THP1 macrophages were stimulated with 100 ng�mL�1

LPS for 10 h. The cells were collected and then split into

two equal fractions. Total RNA was extracted from one

fraction using the RNAiso Plus (Takara) according to the

manufacturer’s instructions while the other fraction was

lysed in ice-cold PBS containing 0.1% NP40 on ice for

15 min. After centrifugation at 500 g for 5 min, the super-

natant was collected as cytoplasmic fraction and the pellets

with an additional wash were considered as nuclear frac-

tions. RNA was then extracted from the nuclear and cyto-

plasm fractions using RNAiso Plus. In order to quantify

gene expression within the different fractions by qPCR, the

total RNA fraction were used to normalize gene expression

across all of the fractions.

RNA immunoprecipitation

RNA immunoprecipitation was performed using Magna

RIP RNA-Binding Protein Immunoprecipitation Kit (Cata-

log # 17-701, Millipore) according to the manufacturer’s

instructions. Anti-p65 (sc-372, Santa Cruz Biotechnology)

or anti-p50 (sc-1190x, Santa Cruz Biotechnology)

2886 FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

MALAT1 regulates the innate immune response G. Zhao et al.

antibodies were used for RIP. Coprecipitated RNAs were

analyzed by qPCR using primers listed in Table S1. Total

RNA (input control) and the isotype control were assayed

simultaneously to show the binding specificity between

MALAT1 and p50/p65.

Statistics

Data are represented as mean � SEM or mean � SD as

noted. Comparisons were performed using two-tailed

paired Student t-test.

Results

MALAT1 is upregulated in LPS-activated

macrophages

It is reported that the LPS-induced transcriptional

response in the human THP1 monocyte cell line is very

similar to peripheral blood mononuclear cell-derived

primary macrophages [23] and therefore, this cell line

has been used extensively to study the response of

monocytes/macrophages to innate ligands, as well as

the role of noncoding RNA in regulating the immune

response [24,25]. Upon treatment with PMA, THP1

cells differentiate into macrophages. Using THP1-

derived macrophages and qPCR, we analyzed expres-

sion patterns of some lncRNAs before and after stimu-

lation with LPS. We observed that MALAT1, a highly

conserved lncRNA in mammalian species, was signifi-

cantly upregulated 10 h after LPS treatment (Fig. 1A).

Similar results were obtained using murine RAW264.7

macrophages (Fig. 1B). Interestingly, MALAT1 was

also upregulated in dendritic cells stimulated by low-

dose LPS (10 ng�mL�1) (Fig. 1C). In THP1 macro-

phages, MALAT1 transcript increased within 1 h of

LPS treatment, reached highest levels at 2 and 8 h,

and then decreased at 14 h poststimulation (Fig. 1D).

These findings imply that in addition to the previously

described role in the development of numerous can-

cers, MALAT1 may play a role in the regulation of

LPS-induced innate immune response.

MALAT1 regulates LPS-induced expression of

proinflammatory cytokines TNFa and IL-6

We next determined whether altered expression of

MALAT1 could regulate the inflammatory response of

macrophages to LPS. For this purpose, THP1 cells

Fig. 1. Analysis of MALAT1 expression in

innate immune cells. (A and B) MALAT1

abundance in macrophages. Following

treatment of THP1 macrophages (A) or

RAW264.7 macrophages (B) with LPS for

10 h, total RNA was extracted and the

level of MALAT1 was measured using

qPCR. Results are mean � SEM of at

least two independent experiments with

duplicate wells. (C) MALAT1 abundance in

bone marrow-derived murine dendritic

cells after treatment with LPS for 6 h.

Results are mean � SEM of two

independent experiments with duplicate

wells. (D) Time course of MALAT1

expression in LPS-treated THP1

macrophages. Results are mean � SEM

of two independent experiments with

duplicate wells. *P < 0.05, **P < 0.01.

2887FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

G. Zhao et al. MALAT1 regulates the innate immune response

were transfected with three siRNAs targeting

MALAT1 and treated with PMA to induce cell differ-

entiation. Although MALAT1 is reported to be

enriched in the nucleus, each of the siMALAT1s sig-

nificantly knocked down MALAT1 expression, with

siMALAT1-1 and siMALAT1-3 being more efficient

than siMALAT1-2 (Fig. 2A). Using qPCR, we mea-

sured mRNA abundance of three well-known proin-

flammatory cytokines, TNFa, IL-1b, and IL-6.

Compared with cells transfected with nonspecific con-

trol siRNA (siNC), knockdown of MALAT1 signifi-

cantly increased the mRNA production of TNFa and

IL-6 following LPS challenge; however, MALAT1

knockdown had no effect on LPS-induced production

of IL-1b mRNA (Fig. 2B). We also determined the

effect of siMALAT1 on cytokine secretion using

ELISA (Fig. 2C). We found that knockdown of

MALAT1 markedly enhanced the level of TNFa and

IL-6 in the supernatant and had no effect on IL-1bsecretion. These data suggest that MALAT1 may

function as a negative regulator of LPS-induced

expression of TNFa and IL-6. Moreover, MALAT1

seems to regulate the LPS-induced inflammatory

response by selectively modulating a subset of inflam-

matory cytokines, as expression of IL-1b, a known

LPS responsive cytokine, did not change in response

to MALAT1 knockdown.

MALAT1 inhibits NF-jB activity

Lipopolysaccharide signals through TLR4 to induce

several distinct signaling pathways in macrophages,

which predominantly converge on the activation of

NF-jB and its target genes, including archetypal

proinflammatory cytokines TNFa, IL-1, and IL-6. We

therefore hypothesized that MALAT1 may modulate

NF-jB function. To test this possibility, we knocked

down MALAT1 in the presence of a NF-jB-dependentluciferase reporter construct. As shown in Fig. 3A,

whereas LPS stimulation increased NF-jB activity in

Fig. 2. Expression of NF-jB-regulated genes in MALAT1 knockdown cells. (A) Knockdown of MALAT1 in LPS-treated THP1 macrophages

with three specific siRNAs. MALAT1 level was assessed by qPCR. A nontargeting siRNA (siNC) was used as control. Results are

mean � SD of one experiment with duplicate wells. **P < 0.01, versus siNC. (B and C) Expression of three NF-jB target genes in THP1

macrophages transfected with control siRNA or MALAT1 siRNAs. The mRNA level of TNFa, IL-6 and IL-1b was quantified by qPCR (B), and

secreted TNFa, IL-6 and IL-1b (C) were quantified by ELISA 10 h after LPS challenge. Results are mean � SD of two independent

experiments with duplicate wells. *P < 0.05, **P < 0.01, versus siNC.

2888 FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

MALAT1 regulates the innate immune response G. Zhao et al.

THP1 macrophages transfected with nontargetting

siRNA control (siNC), NF-jB activity in the macro-

phages transfected with siMALAT1 was much higher.

These observations indicate a negative correlation

between MALAT1 expression and NF-jB activity.

We wonder whether MALAT1 inhibits NF-jBactivity by lowering its abundance. As shown in

Fig. 3B, there was no significant difference in the

mRNA and protein levels of NF-jB subunits p50 and

p65 (RelA) in MALAT1-downregulated THP1 macro-

phages compared to those in the control cells. To

determine whether MALAT1 could change the binding

of p65, a subunit responsible for the transcription acti-

vating potential of jB, to target promoters, we per-

formed ChIP analysis in LPS-activated THP1

macrophages. Knocking down MALAT1 greatly

increased the binding of p65 to TNFa and IL-6 pro-

moters, but not to the IL-1b promoter (Fig. 3C). This

result is consistent with the above mentioned observa-

tion that IL-1b expression did not change in response

to MALAT1 knockdown. Hence, MALAT1 can affect

the ability of p65 to bind to target promoters in a

gene-specific manner.

MALAT1 binds NF-jB in the nucleus

It is well-known that MALAT1 is a nuclear-retained

noncoding RNA [26]; however, several recent publica-

tions report the presence of MALAT1 in the cyto-

plasm to exert its function [27–29], suggesting that

localization of MALAT1 may be cell type-specific. To

determine the subcellular distribution of MALAT1 in

activated macrophages, we isolated nuclear, cytoplas-

mic and total RNA from LPS-stimulated THP1

macrophages. Figure 4A demonstrates the result of

subcellular fractionation, with MALAT1 predomi-

nantly associated with the nucleus.

Although there may be other explanations for the

decreased occupancy of TNFa and IL-6 promoters by

p65 in MALAT1 knockdown cells, the most straight-

forward explanation would be that MALAT1 physi-

cally associates with p65, and occludes it from target

DNA. To test this possibility, we first performed RIP

with the p65 antibody from subcellular extracts of

LPS-stimulated THP1 macrophages. We observed

~sevenfold enrichment of nuclear MALAT1, but not

the cytoplasmic MALAT1, in the anti-p65 immunopre-

cipitates compared with the IgG control (Fig. 4B).

Interestingly, we also observed ~fivefold enrichment of

nuclear MALAT in the anti-p50 immunoprecipitates,

indicating that in the nucleus MALAT1 physically

associates with p65 and p50, two subunits of the pro-

totype NF-jB. To test if MALAT1 associates with the

NF-jB consensus sequence and thus prevents NF-jBfrom binding to its cognate DNA, we performed DNA

pulldown assays using biotinylated oligonucleotides

containing NF-jB-binding sites. It turned out that the

NF-jB consensus DNA probe failed to selectively pull

down MALAT1 from the nuclear extract of LPS-trea-

ted THP1 macrophages (data not shown). Based on

these observations, we conclude that MALAT1 binds

the nuclear p65/p50 heterodimer and thus prevents the

binding of NF-jB to the promoters of a subset of NF-

jB-regulated genes.

LPS-induced MALAT transcription depends on

NF-jB

Given the importance of NF-jB in regulating the tran-

scriptional response to infection, we were interested in

determining whether LPS-induced expression of

MALAT1 is also NF-jB-regulated. We used PDTC,

an inhibitor of NF-jB, to assess the effect of NF-jBinhibition on MALAT1 transcription. As Fig. 5A

shows, PDTC abolished LPS-induced MALAT1

upregulaton in THP1 macrophages. We then per-

formed a computational screen and identified a jBconsensus sequence located at �334 to �325 upstream

of MALAT1 transcription start site. Using a series of

luciferase reporter plasmids containing consecutive

non-overlapping deletions spanning the 50-flankingregion of MALAT1, we found a significant reduction

in promoter activity when the region (�349 to �324)

containing the predicted jB-binding site was deleted

(Fig. 5B). In addition, the LPS-induced luciferase

activity was nearly abolished when the jB-like motif

was point-mutated, confirming the dependence of LPS-

induced MALAT1 upregulaton on NF-jB (Fig. 5C).

Since the deletion and point mutations on the jB-like motif upstream of the MALAT1 transcription

start site had a repressive effect on the luciferase

reporter gene activity, we reasoned that NF-jB is a

key transcription factor necessary for MALAT1 pro-

moter activity. To substantiate the binding of NF-jBto MALAT1 promoter, we performed ChIP with an

antibody against p65 from extracts of THP1 macro-

phages with or without LPS treatment. We observed

significant enrichment of MALAT1 promoter with the

p65 antibody compared with the nonspecific IgG con-

trol antibody (Fig. 5D). Thus, NF-jB directly binds to

the promoter of MALAT1 to activate its transcription.

Discussion

There is accumulating evidence that lncRNAs are

important regulators of physiological and pathological

2889FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

G. Zhao et al. MALAT1 regulates the innate immune response

responses; however, their potential importance in

immunity is only now emerging [19]. During the acti-

vation of the immune response, widespread changes in

the expression of lncRNAs have been recently demon-

strated [30,31]. Although the precise mechanisms by

which most immune-related lncRNAs function remain

poorly understood, some lncRNAs have been found to

play their role through regulating protein-protein inter-

actions or via their ability to base pair with RNA and

DNA [19,20]. In this study, we demonstrate a novel

function of MALAT1, which has been originally iden-

tified as a prognostic parameter for non-small cell lung

cancer, in modulating the production of inflammatory

mediators. Although MALAT1 was expressed in

unstimulated THP1 macrophages, LPS stimulation

enhanced jB-dependent MALAT1 transcription, and

siRNA-mediated knockdown of MALAT1 was

accompanied by a substantial increase in LPS-stimu-

lated production of TNFa and IL-6. Based on our

results, we propose that MALAT1 and NF-jB may

form a nuclear RNA-protein complex and hence the

binding of NF-jB to target promoters is decreased. In

THP1 macrophages, LPS-induced, jB-dependentupregulation of MALAT1 initiates a negative feedback

loop in which the activity of p50/p65 heterodimer, the

predominant form of NF-jB in most cells, is down-

regulated, attenuating the expression of jB-responsivegenes such as TNFa and IL-6 (Fig. 6). This is consis-

tent with the result of in silico analysis that MALAT1

could modulate NF-jB/RelA activity in the context

epithelial–mesenchymal transition [32]. Actually, many

NF-jB target genes, including proteins and micro-

RNA, have been reported to negatively regulate

NF-jB activity at multiple levels of transcription and

Fig. 3. MALAT1 inhibits NF-jB activity. (A) NF-jB reporter activity upon MALAT1 silencing. THP1 macrophages were transfected with

indicated siRNAs and an NF-jB-regulated firefly luciferase reporter plasmid. At 24 h after transfection, cells were stimulated with LPS or

PBS for 10 h. Firefly luciferase activity was analyzed in cell lysates and normalized to the activity of a co-transfected Renilla luciferase

plasmid. Results are mean � SEM of two independent experiments with triplicate wells. (B) Analysis of NF-jB expression upon MALAT1

silencing. THP1 macrophages were transfected with MALAT1 siRNA or scrambled siRNA control (siNC). At 48 h after transfection, cells

were stimulated with LPS for 10 h. Left panel, qPCR analysis of p65 and p50 mRNA levels, with b-actin gene as an internal control. The

data are mean � SEM of two independent experiments with duplicate wells; right panel, the levels of p65 and p50 examined by western

blotting. (C) Binding of p65 to the promoters of three indicated NF-jB-regulated genes. Cells were treated as in (B), and ChIP assays were

performed with IgG and anti-p65 antibody. ChIP values are shown as fold changes of immunoprecipitated promoter fragments over IgG

controls, with siNC ChIP values set as 1. The data are mean � SD of one experiment with duplicate wells.

2890 FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

MALAT1 regulates the innate immune response G. Zhao et al.

post-transcription, and consequently prevent uncon-

trolled and potentially harmful immune responses [33].

An additional emerging concept of NF-jB control is

its regulation through lncRNA that may play their

role via discrete modules that decoy, guide, or scaff-

old other regulatory factors. The identification of

MALAT1, together with recently identified lncRNA

including NKILA, Lethe and PACER, as novel NF-

jB regulators, adds another layer of complexity to the

control of this key transcription factor critical in

immunity and inflammation. The existence of so many

negative regulators and the necessity of each regulator

in the modulation of NF-jB activity imply that combi-

national or synergistic effects among negative regula-

tors are required for full suppression of NF-jBsignaling [7].

MALAT1 is extremely abundant in many human

cell types and is highly conserved across several mam-

malian species underscoring its functional importance

[34]. Previous studies showed that aberrant expression

of MALAT1 correlates with tumor development, pro-

gression, metastasis, and survival in many cancer

types. Besides its oncogenic role in different cancers,

MALAT1 is also involved in other diseases and even

in normal biologic processes such as vessel growth,

synaptogenesis, and myogenesis [28,35,36]. Given its

exceptionally high abundance and strong conservation

in vertebrates, MALAT1 is surprisingly not essential

for life and development in knockout murine models

under normal physiological conditions [34]. Hence,

one central question for the biology of MALAT1 is

to find the right cellular stress and the pathological

or environmental conditions under which MALAT1

becomes essential in vivo [34]. Our work revealed that

under the inflammatory condition triggered by LPS,

MALAT1 is required for tight control of the inflam-

matory response, demonstrating for the first time the

involvement of MALAT1 in regulating innate immu-

nity and inflammation. This finding reminds us of a

report reporting that serum amyloid A3 (SAA3), a

mediator of inflammation, is the only gene whose

expression differed significantly between the wild-type

and MALAT1 knockout mouse liver [37]. Our results

support the hypothesis that the function of MALAT1

depends on the combination of interaction partners in

different cell types. Alternatively, MALAT1 can be

seen as a nuclear or cytoplasmic ‘hub’ for storage

and/or sequestration of distinct RNA-binding proteins

in respective cells that execute its function [34]. As

MALAT1 localizes to hundreds of human genomic

sites most of which are active genes distributed

throughout the genome [38], our understanding of the

function of this classic lncRNA and its mechanism of

action remains far from complete. A systematic iden-

tification of MALAT1-regulated target genes in

monocytes/macrophages that show differential expres-

sion in response to LPS stimulation should provide

information for a better understanding of how

MALAT1 participates in modulating innate immune

responses.

Concerning the role of MALAT1 in regulating the

LPS-activated inflammatory response in macrophages,

there are several questions to which we cannot pro-

vide clear answers. First, although MALAT1 was

Fig. 4. MALAT1 associates with p65 and p50 in the nucleus. (A) Subcellular distribution of MALAT1 in LPS-stimulated THP1 macrophages.

RNA was extracted from total (T), nuclear (N) or cytoplasmic (C) fractions of cells following exposure to 100 ng�mL�1 LPS for 10 h.

MALAT1 present in each fraction was determined by qPCR. The data are mean � SEM of two experiments with duplicate wells. (B)

Interaction between MALAT1 and p65 or p50 revealed by RIP experiments. Nuclear or cytoplasmic extracts of LPS-stimulated THP1

macrophages were immunoprecipitated with control IgG, anti-p65 or anti-p50 antibody, and the complexes were analyzed for the presence

of ACTB mRNA, U6 snRNA or MALAT1 by qPCR. Results are mean � SEM of two independent experiments with duplicate wells.

**P < 0.01.

2891FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

G. Zhao et al. MALAT1 regulates the innate immune response

shown to fulfill its role through interacting with p65/

p50, our data do not rule out the possibility that the

interaction between MALAT1 and NF-jB may be

mediated by other unidentified cellular factors within

the RNA-protein complex. Second, a recent report

described that MALAT1 regulates glucose-induced

up-regulation of inflammatory mediators TNFa and

IL-6 through activation of serum amyloid antigen 3

(SAA3) [39]. We consider that one of the reasons for

the inconsistency in the effect of MALAT1 on TNFaand IL-6 expression may be due to the difference in

cell lines, as functional assays by Puthanveetil et al.

were performed in SAA3-positive human umbilical

vein endothelial cells, whereas the THP1 cells used in

our study is SAA3-negative [40]. We infer that

MALAT1 exerts anti- and proinflammatory effects

through its interactions with various regulatory or

signaling molecules that are differentially expressed in

different cell types. Further study in other cohorts is

needed to fully understand the role of MALAT1 in

balancing TLR signaling and inflammatory responses.

Third, our findings indicate that MALAT1 sup-

pressed LPS-induced mRNA accumulation of TNFaand IL-6 without suppressing IL-1b mRNA produc-

tion. It has been reported that expression of TNFaand IL-6 in monocytes/macrophages is NF-jB-depen-dent because loss of NF-jB activity severely impairs

the production of either cytokine [41,42]; in contrast,

NF-jB activity is not essential for IL-1b production

[43]. As measured by ChIP in this study and in the

work by Ilott et al. [30], IL-1b promoter did not

demonstrate NF-jB-binding. Therefore, the difference

Fig. 5. Lipopolysaccharide-induced MALAT transcription depends on NF-jB. (A) PDTC abrogated LPS-induced MALAT1 upregulation in

THP1 macrophages. Expression of MALAT1 was assayed by qPCR in cells before and after LPS treatment for 10 h, in the presence or

absence of the NF-jB inhibitor PDTC. Results are mean � SEM of two independent experiments with duplicate wells. *P < 0.05,

**P < 0.01, versus PBS or LPS-treated cells without PDTC. (B) Luciferase reporter assays of MALAT1 promoter constructs in THP1

macrophages. Cells were transfected with reporter plasmids containing truncated MALAT1 promoters or the empty vector pGL4.17 and

then treated with LPS for 6 h. Luciferase activities were normalized by Renilla luciferase. Luciferase activities are expressed relative to

the activity of �828/+13, which was given a value of 100%. Results are mean � SEM of two independent experiments with triplicate

wells. (C) NF-jB regulates the MALAT1 promoter. Left panel, MALAT1 promoter luciferase reporter gene constructs with wild-type (wt)

and mutant (mt) jB-like sequences indicated. Right panel, luciferase reporter gene assays for the basal and LPS-stimulated activities of

MALAT1 promoter constructs with wt and mutant jB-binding sites. Luciferase activities were normalized by Renilla luciferase. Results are

mean � SEM of two independent experiments with triplicate wells. **P < 0.01. (D) ChIP analysis of MALAT1 promoter enrichment in

THP1 macrophages. Cells were treated with PBS or LPS for 10 h. After normalization to the input, enrichment is presented as fold over

IgG, which is set as 1. Results are mean � SD of one experiment with duplicate wells. *P <0.05, **P < 0.01.

2892 FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

MALAT1 regulates the innate immune response G. Zhao et al.

in NF-jB dependency might account for the differen-

tial effect of MALAT1 on the three cytokines.

Acknowledgements

This work was supported by Natural Science Founda-

tion of China (81071445).

Author contribution

XM conceived the study, analyzed the data and wrote

the paper. GZ, ZS, DS, and YM performed the experi-

ments, analyzed the data, and contributed to the

preparation of the paper. All authors approved the

contents of this manuscript.

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Supporting information

Additional Supporting Information may be found

online in the supporting information tab for this arti-

cle:Table S1. siRNA and primers used in this study.

2895FEBS Letters 590 (2016) 2884–2895 ª 2016 Federation of European Biochemical Societies

G. Zhao et al. MALAT1 regulates the innate immune response

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