Life Sciences 266 (2021) 118895

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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Review article

Long non-coding RNAs as the regulators and targets of macrophage

M2 polarization
Rong Dong a, Bo Zhang b, Biqin Tan a, Nengming Lin a, b, *
a
Department of Clinical Pharmacy, Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Affiliated Hangzhou First People’s
Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
b
Translational Medicine Research Center, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 31006, China

A R T I C L E I N F O A B S T R A C T

Keywords: Macrophages are immune cells with high heterogeneity and plasticity. M2 polarization is one extreme of the
Long non-coding RNAs well-established phenotypes of macrophage polarization, and involves in diverse biological processes. The po­
M2 polarization larization process is initiated at the command of numerous components. Long non-coding RNAs (lncRNAs) are
Macrophages
RNAs longer than 200 nucleotides with limited protein-coding capacity. Recent studies have revealed a newly
Antisense oligonucleotides
found subset of lncRNAs engaged in the M2 polarization and their potent and multifunctional roles in developing
CRISPR interference
diseases. By interfering with specific signaling pathways and altering the active mode, acting as the sponges of
microRNAs or decoys of transcription factors, lncRNAs prompted macrophages to an M2 phenotype. Further,
lncRNAs can bind to the genome to regulate the chromatin dynamics or work as a platform for protein complexes
tether. Exosomal lncRNAs can also orchestrate the polarization in a paracrine way. To make it easier to interpret
the roles of lncRNAs in the M2 polarization, we review the reported lncRNAs according to the underlying
mechanisms. Moreover, we discuss the possibilities of targeting macrophages’ M2 polarization using the oli­
gonucleotides drugs or clustered regularly interspaced palindromic repeats (CRISPR) technologies to provoke
wisdom on the therapeutic strategies.

1. Introduction interleukin-13 (IL-13) may induce alternatively activated macrophages,

Macrophages are crucial antigen-presenting cells with phenotypic
and functional plasticity in the immune system [1]. Versatile polarized
macrophages could exert different functions depending on the circum­
stances [2,3]. Macrophages stimulated by bacterial lipopolysaccharide
(LPS) or interferon-γ (IFN-γ) are the so-called classically activated
macrophages, or M1 macrophages, which promote the production of
pro-inflammatory factors [4–6]. However, interleukin-4 (IL-4) or
also known as M2 macrophages, which are involved in the anti-
inflammatory factors’ generation and tissue repair [7]. Usually, M2
macrophage can promote inflammation reduction, and wound healing
[8,9]. When infiltrated into the tumor microenvironment, macrophages
are often called tumor-associated macrophages (TAMs), which are often
considered to be synonymous with M2 like and can promote tumori­
genesis, proliferation, metastasis, and angiogenesis [10–12]. Targeting
the M2 macrophages or the regulators of M2 polarization is considered a

Abbreviations: LncRNAs, long non-coding RNAs; LPS, lipopolysaccharide; IFN-γ, interferon-γ; IL-4, interleukin-4; IL-13, interleukin-13; TAMs, tumor associated
macrophages; JAK2, Janus Kinase 2; STAT, signal transducer and activator of transcription; SNHG20, small nucleolar RNA host gene 20; EMT, epithelial mesen­
chymal transition; HMGB1, high mobility group box 1 protein; USP11, ubiquitin specific protease 11; TLRs, toll like receptors; FISH, fluorescence in situ hybridi­
zation; MIR155HG, miRNA 155 host gene; COPD, chronic obstructive pulmonary disease; NF-κB, nuclear factor kappa-B; IL-10, interleukin-10; NSCLC, non-small cell
lung cancer; NECAB3, N-terminal EF-hand calcium binding protein 3; CCAT1: HIF-1α, hypoxia inducible factor 1 subunit alpha; PRC2, polycomb-group proteins; T-
UCRs, transcribed ultra-conserved RNAs; β-TrCP, beta-transducin repeat containing protein; GAS5, growth arrest-specific 5; IRF4, interferon regulatory factor 4;
EZH2, enhancer of zeste homolog 2; H3K27me3, trimethylation of lysine 27 on histone 3; SOCS3, suppressor of cytokine signaling 3; HCC, hepatocellular carcinoma;
MALAT1, metastasis associated in lung adenocarcinoma; COX-2, cyclooxygenase-2; ASOs, antisense oligonucleotides; CRISPRs, clustered regularly interspaced
palindromic repeats; crRNA, CRISPR-derived RNA; tracrRNA, trans-activating RNA; sgRNA, singleguide RNA; DETECTR, DNA endonuclease-targeted CRISPR trans
reporter; PAM, protospacer adjacent motif; PPAR-γ, peroxisome proliferators-activated receptor-γ.
* Corresponding author at: Room 207, No. 5 Building, Department of Clinical Pharmacy, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of
Medicine, Hangzhou, China.
E-mail address: lnm1013@zju.edu.cn (N. Lin).

https://doi.org/10.1016/j.lfs.2020.118895
Received 9 September 2020; Received in revised form 16 November 2020; Accepted 4 December 2020
Available online 10 December 2020
0024-3205/© 2020 Elsevier Inc. All rights reserved.
R. Dong et al.
new approach to conquer the immunological diseases [13].
Precise regulation of the polarization of macrophages is a critical
step to arouse the specific function of the immune system [14]. The
majority of studies have focused on the coding genes, transcription
factors, and pathways in these processes. Studies with macrophages
from the IL-4 receptor-deficient mice were found to lack M2 macrophage
development in mouse models of Th2 cell-mediated inflammation [15].
The phenotype that defines M2 is characterized by the increased pro­
duction of IL-10, TGF- TGF-β, Arg-1, Fizz1, and CCL18. These charac­
teristics are promoted by the dimerization of IL-4Rα and subsequent
JAK-mediated tyrosine phosphorylation of STAT6 [16]. On the other
hand, the STAT6 can bind to PPAR-γ, a master regulator of lipid meta­
bolism in macrophages, and is known to inhibit the pro-inflammatory
gene expression and act as a cofactor in PPAR-γ mediated gene regula­
tion in metabolic dysfunction and diseases [17]. Overwhelming evi­
dence supports the role of the PI3K-Akt signaling pathway in M2
polarization. PI3K generates phosphatidylinositol-3,4,5-triphosphate,
which activates the downstream protein kinase B- Akt. Akt has three
distinct isoforms: Akt1, 2, and 3. Studies utilizing Akt isoforms-specific
deficient mice revealed that Akt1 promotes the polarization of M2
macrophages and inhibits the polarization of M1 macrophages, while
Akt2 vice versa [18]. MicroRNA-155 (miR-155) and C/EBPβ play a vital
role in regulating Akt-dependent macrophage polarization. Akt2 en­
hances the expression of miR-155, which down-regulates the expression
of C/EBPβ, and ultimately promotes the polarization of M1 macrophages
[19].
Comparatively, less is known about the non-coding RNAs’ functions
of, especially the long non-coding RNAs (lncRNAs) [20]. LncRNAs are an
array of RNAs longer than 200 nucleotides with limited protein-coding
potential [21–23]. Thanks to the advances of the next generation
sequencing technology, a series of lncRNAs are annotated and func­
tionally analyzed well in the differentiation and maturation of various
immune cells [24–26]. LncRNA-Morrbid was found to control the sur­
vival of short-lived myeloid cells via the cis-regulation of Bcl2l11
expression [27]. Lnc-DC promotes the dendritic cells by the C terminus
of STAT3 to prevent the dephosphorylation of STAT3 Y705 by SHP1
[28]. The lnc-MC regulates the differentiation of monocyte to macro­
phage to some extent [29]. Systematic research has suggested that
several lncRNAs are dynamically changing during the polarization of
macrophages. But the annotation of lncRNAs is still far from clear, and as
such, no existing comprehensive study exists has reviewed the role of
lncRNAs specifically involved in the process of macrophage M2 polari­
zation. Consequentially, here we review the recent advances in eluci­
dation of various lncRNAs and their mechanisms within the M2
polarization. In addition, we discuss the possibility of applying antisense
nucleic acid drugs to intervene in the process to cure related diseases.
2. Overview of lncRNAs
Comprehensive analyses of the human genome have shown that
although large numbers of RNA transcripts are transcribed into proteins,
they only take up a very small portion of the transcripts. The rest of the
transcripts are non-coding RNAs, including miRNAs, long non-coding
RNAs, piRNAs, circRNAs, etc. [30]. Long non-coding RNAs are always
longer than 200 nt with little ability to encode proteins or polypeptides.
Unlike the message RNAs, lncRNAs may have polyadenylic acid tails or
not. Compared with the protein-coding genes, they have higher tissue
and organ specificity but less low conserved sequence among species.
There are several ways to classify lncRNAs, one of the simple ways is
based on the genomic level organization [31]. In this aspect, they are
divided into five groups: (1) sense lncRNAs, the transcription direction
of this type of lncRNAs is the same as that of the neighborhood mRNAs.
(2) Antisense lncRNAs are those whose transcription direction is oppo­
site to that of the protein-coding genes. (3) Bidirectional lncRNAs can be
transcribed from the same or the opposite directions to the adjacent
mRNA transcription. (4) Intergenic lncRNAs are transcribed between
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Life Sciences 266 (2021) 118895
two protein-coding genes. (5) Intronic lncRNAs are transcribed from the
intron region of the genome. As the research goes deepen, we must
realize that lncRNAs can function independently of their genome loci’s
DNA sequence.
Herein we review our current understanding of lncRNA functions.
Firstly, lncRNAs can interfere with specific signaling pathways and alter
the active mode [32]. They can also act as the sponges of microRNAs,
decoys of transcription factors, or transcriptional coactivators or co­
repressors [33]. Third, they can bind to the genome to regulate chro­
matin dynamics, histone modifications, methylation or downstream
gene expression [34–37]. Finally, lncRNAs could alter the localization,
transportation of proteins, or the protein complex formation.
3. LncRNAs in M2 polarization
3.1. Signaling lncRNAs
LncRNAs are pivotal regulators in the macrophage polarization
processes in response to intracellular or extracellular stimuli (Table 1).
The commonly held view is that M2 polarization is driven by Th2 cy­
tokines or chemokines, such as IL-4 or IL-13 [12]. These cues are thought
to provoke the level change of lncRNAs and activate the JAK2-STAT6
pathway, MAPK-ERK/MEK/JNK pathway, and PI3K-Akt pathway to
promote the M2 polarization [14]. Many lncRNAs have been docu­
mented to regulate the polarization in this manner (Fig. 1a).
One of these lncRNAs is small nucleolar RNA host gene 20
(SNHG20), a 2183 bp transcript originated from the chromosome
17q25.2. It was first reported in human hepatocellular carcinoma and
then expanded to various malignancies [38]. SNHG20 could control the
proliferation via the cell cycle-associated genes, e.g., Cyclin A1, to
promote the metastasis via the EMT signaling pathway [39]. Recently,
SNHG20 was suggested to be highly expressed in Kupffer cells, the
resident macrophages in the liver, during nonalcoholic fatty liver dis­
ease progression to hepatocellular carcinoma [40]. Gain and loss of
function study of SNHG20 in Kupffer cells revealed that M2 polarization
induced and M1 polarization was deduced in RAW264.7 cells, respec­
tively. Increased phosphorylation of STAT6, a classical mediator of M2
polarization, was observed when SNHG20 was overexpressed. STAT6
inhibitor AS1517499 efficiently decreased the promotion of M2 polari­
zation [41].
Our previous work also determined another novel murine lncRNA,
5730422e09Rik, which we referred to as lncRNA MM2P, was involved
in the M2 polarization [42]. MM2P is a 1715 bp transcript from chro­
mosome 5, between gene HMGB1 and USP11 [43,44]. When we started
the mechanism study, we assumed MM2P might be associated with
HMGB1 protein, since HMGB1 was a key regulator in initiating and
maintaining of chronic inflammation through TLRs signaling pathways
and chromosomal binding. But the FISH assay revealed MM2P was
predominantly localized in the cytosol. Knockdown MM2P affected the
phosphorylation of STAT6 but not the upstream kinase JAK2. When
applying the chemical agent Na3VO4, it can partially reverse the
dephosphorylation of STAT6 induced by MM2P. Therefore, we supposed
MM2P has participated in the dephosphorylation of STAT6, but the
further remains exclusive. The biological analysis revealed that MM2P
promoted the tumorigenesis, angiogenesis of osteosarcoma.
MiRNA-155 is a micro non-coding RNA involved in immunity,
inflammation and tumors [45]. Its host gene was a long non-coding RNA
named MIR155HG [46]. Recently, MIR155HG was found to regulate
macrophages’ polarization in chronic obstructive pulmonary disease
(COPD) patients [47]. Overexpression of MIR155HG could increase the
M1 but decrease the M2 polarization. Luciferase assay revealed that NF-
κB protein p65 increased the expression of MIR155HG through binding
to the upstream of the transcription stat site of MIR155HG.
R. Dong et al. Life Sciences 266 (2021) 118895

Table 1
Summary of lncRNAs involved in the M2 polarization and mechanisms.
lncRNA Mechanism of action and Disease Cell type Ref.
function
SNHG20 Induce M2 polarization Nonalcoholic RAW264.7, [40]
through STAT6 fatty liver Liver
disease to nonparenchymal
hepatocellular cells
carcinoma
MIR155HG Knockdown of MIR-155HG Chronic PBMCs [47]
(MIR155 inhibits the polarization of obstructive
host gene) M1 macrophages and pulmonary
increase M2 macrophage disease
polarization (COPD)
MM2P Promote M2 polarization Osteosarcoma RAW264.7, [42]
through the BMDMs
dephosphorylation of STAT6
KCNQ1OT1 Function as a decoy of Aseptic RAW264.7, [50]
miR-21a-5p, regulate IL-10 loosening after BMDMs
expression, and ameliorate total joint
particle-induced replacement
NIFK-AS1 Inhibit M2 polarization via Endometrial PBMCs, TAMs, [51]
targeting miR-146a, thereby cancer THP-1
reducing the
estrogen-induced
proliferation, migration an d
invasion of endometrial
cancer cells
CCAT1 CCAT1 knockdown promote Prostate cancer RAW264.7, [52]
M2 polarization by PBMCs
upregulating miR-148a (not
a sponge)
RP11-361F Act as a competing Osteosarcoma RAW264.7 [54]
15.2 endogenous RNA against
miR-30c-5p and upregulate
CPEB4 expression,
promote proliferation,
migration, invasion and
M2-like polarization in
osteosarcoma
GNAS-AS1 Promote M2 polarization NSCLC THP-1, PBMCs [55]
and NSCLC progression via
inhibiting miR-4319, which
could target N-terminal

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R. Dong et al. Life Sciences 266 (2021) 118895

EF-hand calcium binding

protein 3
linc-p21 Knockdown of lincRNA-p21 Breast cancer TAMs [56]
promote MDM2 to
antagonize p53 function,
tumor cell apoptosis,
migration, and invasion
uc.306 May target gene BTRC and Hepatocellular U937 [57]
involved in Wnt signaling carcinoma
pathway
GAS5 Promote the M1 Diabetic PBMCs [59]
polarization by upregulating wounds
STAT1, reduction of GAS5 RAW264.7
enhance the transition of Childhood Microglia from
M1 macrophages to M2 pneumonia mice
macrophages Multiple
act as a sponge for sclerosis
miR-455-5p to facilitate
SOCS3 expression
Suppress transcription of
TRF4 by recruiting the
PRC2 to inhibit microglial
M2 polarization
MALAT1 Promote the expression of Pre-eclampsia MSCs, THP-1 [66]
VEGF not only through
affecting the promoter but
also the miRNAs and Atherosclerosis
regulate the proliferation,
angiogenesis and
immunosuppressive
properties of MSCs
Exosomal MALAT1 derived
from oxLDL-treated
endothelial cells promoted
M2 polarization
LINC00662 Physically bind miR-15a, Hepatocellular THP-1 [65]
miR-16, miR-107, activate carcinoma
Wnt/β-catenin signaling in a
paracrine manner
CASC2c Interact with and repress Glioblastoma THP-1, BMDMs [68]
miR-338-3P multiforme
COX-2 Inhibit immune evasion and Hepatocellular RAW264.7 [69]
tumor growth by inhibiting carcinoma
the polarization of M2
macrophages
RPPH1 CRC cells-derived Colorectal PBMCs [71]
exosomes transport RPPH1 cancer
into macrophages and
mediate M2 polarization
TUC339 Required for IL-4 Hepatocellular THP-1, [70]
macrophages polarization carcinoma

RAW264.7: murine macrophage/monocyte like cell linage, originating from Abelson leukemia virus
transformed cell linage derived from BALB/c mice; PBMCs: peripheral blood mononuclear cells;
BMDMs: bone marrow-derived macrophages; THP-1: human acute monocytic leukemia cell line;
TAMs: tumor associated macrophages; U937: human histiocytic lymphoma cells; MSCs: mesen­
chymal stem cells.
Each of the color shading represents a subgroup of lncRNAs classified according to the lncRNA’
function. The lncRNAs in blue shading are signaling lncRNAs. The lncRNAs in pink shading are

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R. Dong et al.
decoy lncRNAs. The lncRNAs in yellow shading are guide lncRNAs. The lncRNAs in pink shading are
scoffold lncRNAs. The last part are exosomal lncRNAs.
3.2. Decoy lncRNAs
Another way for lncRNAs to participate in macrophage polarization
is to compete with other endogenous molecules [48,49]. They can
sponge miRNAs or decoy transcription factors to change protein tran­
scription or degradation. This kind of interference alters the polarization
state and networks of macrophages as well (Fig. 1b).
LncRNA KCNQ1OT1 is a bidirectional antisense long non-coding
transcript locus on chromosome 12p15.5. It was reported to be a
sponge of miR-370 in glioma tissues, a competing endogenous RNA of
miR-701-3p in osteoblast. The report showed that KCNQ1OT1 decoyed
the miR-21a-5p in macrophages to regulate the IL-10 expression, further
promoted M2 polarization, and ameliorated osteolysis [50].
Zhou et al. reported that lncRNA NIFK-AS1, a 1267 bp-long antisense
RNA locus on the chromosome 2q14.3, could regulate the M2 polari­
zation in endometrial cancer recently [51]. Bioinformatics analysis
revealed that NIFK-AS1 could target miR-146a and thus promote the
expression of Notch1 protein. LncRNA GNAS-AS1 is another antisense
long non-coding RNA from the chromosome 20q13.32. It was reported
to increase in the TAMs from NSCLC patients and promote the NSCLC
cell proliferation, migration and invasion by directly competitively
binding of miR-431 with protein NECAB3.
Studies have reported other lncRNAs that could promote M2 polar­
ization through miRNAs, such as lncRNA CCAT1 [52,53]. However, it
did not confirm the sponge or decoy role of CCAT1; therefore, the
mechanism may need further work. Rp11-361F15.2 was also a lncRNA
that reported to promote the M2 polarization, as it was a competing
ceRNA against miR-30c-5p in the osteosarcoma [54]. In NSCLC speci­
mens, GNAS-AS1 was found to promote M2 polarization via inhibiting
miR-4319, which could target the N-terminal EF-hand calcium-binding
protein3 [55].
3.3. Guide lncRNAs
Guide lncRNAs could bind to enzymes or protein complexes to
activate downstream cascades. This kind of lncRNAs is seldom reported
in macrophage polarization since the precise biological system associ­
ated with macrophage polarization is still being studied. Only a few
were supposed to regulate the polarization in this way (Fig. 1c).
LincRNA-p21 was about 3000 bp in length locus on the upstream of
protein p21, a cell-cycle and proliferation regulator, and regulated the
p21 level. Other reports suggested that linc-p21 could interact with
proteins like MDM2, p53, HIF-1α, PRC2. A recent study reported that the
linc-p21-MDM2-p53 axis was involved in the TAMs. LincRNA-p21 pro­
moted the function of TAMs by preventing the combination of MDM2
and p53. Once lincRNA-p21 was knockdown, MDM2 stayed in the nu­
cleus and NF-κB-STAT3 was activated to reeducate the TAMs to an M1
phenotype [56].
Transcribed ultra-conserved RNAs (T-UCRs) are a novel subset of
long non-coding RNAs that are completely conserved across disparate
taxa. Increasing evidence showed that T-UCRs also regulate the
macrophage polarization in disease development. T-UCR uc.306 was
one of the top differentially expressed T-UCRs in the M2 polarization
status [57]. Target gene prediction revised that uc.306 might be the E3
ligase of β-TrCP, an E3 ligase of β-catenin. β-Catenin is one of the key
regulators in the Wnt signaling pathway activation. Wnt signaling
pathway can manipulate the activation status of macrophages. Luo and
colleagues found that uc.306 might be upregulated in M1 macrophages
and lay a low profile in HBV-related HCC.
3.4. Scaffold lncRNAs
Scaffold lncRNAs can interact with various protein complexes and
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Life Sciences 266 (2021) 118895
direct them to target genomic location and gene promoter.
LncRNA-GAS5 was suggested as a 630 bp long tumor suppressor
lncRNA locus on the chromosome 1q25. It participated in the prolifer­
ation, apoptosis, cell migration and cellular metabolism to promote the
malignancy [58]. As research continues to deepen, the role of GAS5 in
the immune system has become clear. Zhao and colleagues suggested
that GAS5 could suppress IRF4 transcription by binding PRC2 to inhibit
M2 polarization in the autoimmune encephalomyelitis animal model of
multiple sclerosis [59]. Binding between EZH2 and IRF4 promoter,
H3K27me3 and IRF4 promoter was attenuated by GAS5 (Fig. 1d).
Although we considered GAS5 a scaffold lncRNA, it was reported to
promote M1 polarization by upregulating STAT1 as a signaling lncRNA
in the diabetic wounds healing and acted as a sponge for miR-455-5p to
facilitate SOCS3 expression in the childhood pneumonia [60,61].
3.5. Exosomal lncRNAs
As previously described, lncRNAs might parent from macrophages’
genome and they manipulated the polarization process. As the research
continues to deepen, lncRNAs from the nearby cells also can interfere
with the macrophage polarization in a paracrine way [62].
Exosomes are small extracellular vesicles released from cell mem­
branes with a size of about 30–100 nm. They can be biological depots
carrying proteins, mRNAs or lncRNAs in various fluids and help
communicate between different cells [63]. Exosomal lncRNAs are
implicated in the M2 polarization as well (Fig. 1e).
Linc00662 was a 2085 bp long transcript originated from chromo­
some 19q11. It could physically bind miR-15a, miR-16, and miR-107,
which target WNT3A [64]. Tian and colleagues found that linc00662
could promote the HCC progression in an autocrine manner and pro­
mote the TAMs to a M2 polarization via the exosomal linc00662 in a
paracrine manner. Both biological effects were promoted by activating
the Wnt/β-catenin signaling pathway [65].
Other reported exosomal lncRNAs included MALAT1 in pre-
eclampsia and atherosclerosis [66,67], CASC2c in glioblastoma multi­
forme [68], COX-2 and TUC339 in hepatocellular carcinoma [69,70],
and RPPH1 in colorectal cancer [71]. They were all reported to promote
the M2 polarization by secretory exosomes between different cell
populations.
4. Therapeutic strategies targeting the macrophage polarization
Macrophages are the first line of the immune system. Abundant
macrophages infiltrated in the milieu may elicit extensive responses
dependent on the polarization state. Non-coding RNAs take up about a
significant portion of the whole genome. They decline the exponentially
important role in the macrophage polarization processes and are rapidly
emerging as novel therapeutic targets because of their abundantly
expressed properties and specific expression patterns [72–75]. We
would like to discuss two novel therapeutic strategies other than the
traditional chemical agents targeting the macrophage polarization
pathways.
4.1. Antisense oligonucleotides interference
Small interference RNA technologies are classical means to study the
gene function according to the lab’s base complementary principle in
the lab [76,77]. Now patients have the opportunities to benefit from
these technologies as the applications go from bench to bedside [78–80].
RNA interference-mediated TMPRSS6 mRNA degradation improved the
erythropoiesis and anemia in animal models, and later SLN124, a
GalNAc-siRNA conjugate targeting TMPRSS6, has been granted as an
orphan drug by the European medicines agency [81,82]. However,
R. Dong et al. Life Sciences 266 (2021) 118895

Fig. 1. Involvement and regulatory mechanisms of lncRNAs in M2 macrophage polarization. (a) lncRNAs can interfere with specific signaling pathways and alter the
active mode. (b) lncRNAs can act as the sponges of microRNAs, decoys of transcription factors or transcriptional coactivators or corepressors. (c) lncRNAs can bind to
the genome to regulate the chromatin dynamics, histone modifications, methylation or downstream gene expression. (d) lncRNAs could alter the localization,
transportation of proteins or the formation of the protein complex. (e) lncRNAs from the nearby cells also can interfere with the macrophage polarization in a
paracrine way.

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R. Dong et al.
unlike protein-coding genes, lncRNAs may locate in the nucleus, which
is not easy to reach for siRNAs. In addition, the knockdown efficiency of
siRNAs may not last long since the siRNAs are vulnerable to the RNases.
Antisense oligonucleotides (ASOs) are synthetic single-stranded
nucleic acids complementary to their target and trigger RNase H-
mediated degradation [83,84]. To avoid the disadvantages of siRNAs,
ASOs must meet two criteria [85]. First, they should not be degraded by
endogenous nucleases to be sent to target cells to produce the most
significant effect. Presently, viral vectors are used to transfer to cells
[86,87]. But the side effects of viral vectors prompted researchers to
explore and develop non-viral transfer systems considering the safety
and clinical application [88]. In addition, the antisense nucleic acid
molecules must be able to approach the target tissue at a specific con­
centration and penetrate the cell membrane into the cell to produce a
therapeutic effect [89]. Thus, various chemical modifications and
structural modifications to ASOs have been done to ensure their efficacy
[90]. The first generation is to modify the backbone with phosphate;
thus, the phosphorothioates are the most widely used of this kind. Later,
diverse chemical groups, like alkyl modifications at the 2′ position of the
ribose and locked nucleic acids, were introduced into the structure to
improve the binding affinity, enzyme resistance and pharmacokinetics
profile.
Gong et al. constructed specific ASOs targeted lncRNA MALAT1 to
control breast cancer’s metastasis [91]. The ASOs were packed with a
nucleus-targeting TAT peptide in Au nanoparticles to downregulate the
expression of MALAT1. The TAT peptide could lead the ASOs into the
nuclear, and Au nanoparticles could defeat the endonucleases and
exonucleases. These kinds of modifications of ASOs reveal the possibil­
ities to target lncRNAs to cure diseases.
4.2. Clustered regularly interspaced palindromic repeats interference
Gene-editing technology has reached its peak by unmasking the role
of the clustered regularly interspaced palindromic repeats (CRISPRs)
and its Cas9 nuclease enzyme in the bacteria. CRISPR-Cas9 system works
as a prokaryotic adaptive immune system to purge phages and foreign
plasmids [92]. The crRNA (CRISPR-derived RNA) can bind to the
tracrRNA (trans-activating RNA) through base pairing to form the
tracrRNA/crRNA complex, which guides the nuclease cas9 protein to the
target site to cut double-stranded DNA [93]. The cas-mediated double-
strain break will next activate the homology-directed repair or non-
homologous end joining pathway. By artificially designing these two
types of RNAs, these can be transformed into a single-guide RNA
(sgRNA), which is enough to guide cas9 dependent cleavage of DNA
[94]. Now it has been used as a potent gene manipulation tool to cure
diseases [95].
CRISPR interference could help screen genome-wide coding and non-
coding genes functions, including lncRNAs [96]. S. John et al. recently
used CRISPRi technology to screen the potential targets of lncRNAs in
the glioblastoma cells, and nine candidates were found to be relevant to
the sensitivity of glioma cells to radiation therapy [97]. ASOs were
applied to this study to reveal the role of lncGRS-1, which could selec­
tively inhibit the glioma cells’ growth and resistance to radiation.
Systematically studies continue, more cas-nucleases were found to
expand the precise gene editing tools. Cas12a was found to cleave single-
stranded DNA as well as double-stranded DNA [98]. Combining cas12a,
a method named DETECTR (DNA endonuclease-targeted CRISPR trans
reporter) was developed to detect the human papillomavirus in patient
samples [99]. Cas13a can also be engineered for RNA knockdown and
binding [100,101]. Cas14 can bind and cleave single-stranded DNA in­
dependent of PAM sequences [102]. All these gene editing technologies
are revolutionary weapons which can be applied to target the lncRNAs.
4.3. Oligonucleotide drugs in macrophages
To date, an increasing number of lncRNAs in the M2 polarization
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Life Sciences 266 (2021) 118895
have been discovered. Although currently available technologies such as
small RNA interference and CRISPR-based libraries screen make it easier
to discover and target the lncRNAs, there are seldom oligonucleotide
drugs specifically targeting lncRNAs modulating M2 polarization. While
some researches based on oligonucleotide therapeutics may shed light
on this problem.
AZD9150 is a 16-nucleotide STAT3 ASO. It is designed to target and
indirectly downregulate the expression of STAT3 mRNA and has pre­
sented its efficacy in refractory/relapsed lymphoma and DLBCL [103].
As the JAK/STAT signaling pathway is a critical regulator in the
macrophage polarization, studies with the connection between anti-
cancer efficacy and the states of peripheral blood mononuclear cells
would be further investigated. Other ASO drugs include an antisense
oligonucleotide drug on lung fibrosis. NF-κB is a crucial nuclear tran­
scription factor. It participates in the inflammatory response, immune
response, and cell apoptosis. The over-activation of NF-κB is found in
many human diseases such as rheumatoid arthritis, inflammatory dis­
eases in the heart and brain. H. Saitoh et al. have revealed the effect of an
antisense oligonucleotide drug to the p65 subunit of NF-κB in the ARDS
mouse model [104,105]. The suppression of NF-κB in the alveolar
macrophages resulted in the decreased secretion of IL-8. Yasunori et al.
also reported the successful delivery of locked nucleic acid-containing
antisense oligonucleotides in the alveolar macrophages [106].
Genome-wide CRISPR screen has been used to identify functional
non-coding RNAs in cell differentiation and development. Phagocytosis
is a significant skill for the macrophage to neutralize and terminate
foreign pathogens. CRISPR-based library screen has revealed multiple
novel genes in the phagocytic processes. F. Imperatore et al. revealed
that CRISPR/Cas9 mediated deletion of SIRT1 impacts the steady-state
and self-renewal in alveolar and peritoneal macrophages [107].
Knockout or the CRISPRi in-vivo models of lncRNA-Cox2 revealed that
the altered expression of inflammatory genes in lncRNA-Cox2 deficient
macrophages and murine tissues confirmed its trans- and cis- manipu­
lation of the neighbor gene Ptgs2 and its drastic influence in the immune
signaling axes [108]. Experimental studies also confirmed that lncRNA-
ANRIL is capable of forming circANRIL. CRISPR-based deletion of cir­
cANRIL isoforms controls the cell proliferation and apoptosis in mac­
rophages in atherosclerosis [109].
5. Conclusions
Macrophages are in constant changes with two extreme polarization
states, respectively M1 and M2 macrophages. The M2 macrophages
were promoters in various diseases. Distinguishing the pivotal regula­
tors of macrophage polarization may be a significant step to cure dis­
eases. Though the studies of lncRNAs are still in its infancy, increasing
numbers of lncRNAs were investigated in the macrophages’ M2 polari­
zation process [110,111]. Their multifunctional capabilities and het­
erogeneous working mechanisms make them potential targets for the
manipulation of diseases [112].
Gene therapy has been an attractive and alternative approach to cure
diseases, especially the monogenic human genetic diseases, due to gene-
editing technologies’ breakthrough. Discrepancies between the RNAi-
and CRISPRi-based therapies make it comparatively easy to screen and
target the lncRNAs in the complicated biological reactions in versatile
modality. Oligonucleotide drugs can be used to modulate the non-
coding gene expression, as well as the protein-coding genes [83,113].
They work through the complementary Watson-Crick base pairing
principle and so interrogate the presented target. Though the cross-
species conservation of lncRNAs is relatively low, the tissue-specific
lncRNA expression shows strong conservation across species, and
neighborhood gene locus is quite conserved. The length of ASOs
partially improves their specificity because 16 to 20 nucleotides can
uniquely bind to the target. After binding to the target RNA, they can
regulate the RNA by promoting the fragmentation or degradation of
target RNA or sterically blocking the target [114,115]. As of January
R. Dong et al.
2020, ten oligonucleotide drugs have received approval from the FDA,
and over 100 oligonucleotides drugs are in clinical trials [116]. Five of
the ten approved drugs target the liver organ since it is pivotal in drug
metabolism and detoxification [117]. Kupffer cells are resident macro­
phages in the liver and account for most uptake of the ASO drugs. The
knockout of scavenger receptor A in Kupffer cells results in about 25%
reduction of the accumulation and a 50% decrease of ASO drugs [118].
Targeting macrophages and targeting the lncRNAs using the nucleic
acid-based therapeutics may hold therapeutic potential.
In conclusion, lncRNAs exhibit tremendous effects on the macro­
phage differentiation and polarization, providing other druggable tar­
gets for the interference of macrophage engaged disorders.
Funding
This work was supported by the Zhejiang Provincial Natural Science
Foundation (grant number LQ19H160012 to Rong Dong).
CRediT authorship contribution statement
Conceptualization, Rong Dong. and Biqin Tan.; methodology, Rong
Dong.; investigation, Rong Dong.; resources, Biqin Tan.; wri­
ting—original draft preparation, Rong Dong.; writing—review and
editing, Biqin Tan and Bo Zhang.; visualization, Rong Dong.; supervi­
sion, Nengming Lin.; project administration, Nengming Lin.; funding
acquisition, Rong Dong. All authors have read and agreed to the pub­
lished version of the manuscript.
Declaration of competing interest
The authors declare that they have no competing interests.
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