Biomedicine & Pharmacotherapy 123 (2020) 109716

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Review

Advances in the study of exosomal lncRNAs in tumors and the selection of T

research methods
Yishi Wu, Yuhui Wang, Min Wei, Xiao Han, Tianmin Xu*, Manhua Cui*
Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, 130041, Jilin, China

A R T I C LE I N FO A B S T R A C T

Keywords: Exosomes are endosome-derived extracellular vesicles that are released upon the fusion of multivesicular bodies
Exosomes with the plasma membrane. These vesicles contain proteins, lipids, and nucleic acids and are found in various
LncRNA human body fluids. Exosomes can transfer bioactive molecules to nearby or distant recipient cells, thereby
Research methods affecting their function. Recently, exosomes have gained importance as a medium of communication between
Tumor
tumor cells. An increasing number of studies have found that non-coding RNAs in tumor cell-derived exosomes
Chemoresistance
can regulate tumor microenvironments, inhibit immune cell function, promote the growth and invasion of tumor
Biomarkers
cells, and impart resistance to chemicals in tumor cells. In this review, we focus on the effects of exosomal long
non-coding RNAs (lncRNAs) on tumors. As exosomes and their parent cells have similar biological characteristics
and coated lncRNAs can exist stably in vivo without being degraded by RNases, exosomal lncRNAs have emerged
as novel non-invasive tumor biomarkers for use in the early diagnosis and evaluation of prognosis of tumors.
Advancements in the field have led to the development of a variety of techniques in exosomal non-coding RNA
research. Currently, most methods include the separation and purification of exosomes, followed by RNA ex-
traction, reverse transcription, and subsequent analyses; thus, these processes are very tedious and vulnerable to
contamination and could lead to inaccurate and inconsistent results. Thus, there has been an increase in the
development of detection methods for exosomal RNAs. Here, we discuss the existing research methods, their
advantages and disadvantages, and a few new techniques.

1. Introduction
Extracellular vesicles (EVs) are nanometer-sized lipid bilayer ve-
sicles. According to their size and buoyant densities, EVs can be clas-
sified as exosomes, ectosomes, or microvesicles and apoptotic bodies
(ABs) [1]. Initially, these extracellular vesicles were considered waste
bodies excreted by cells. However, with advances in research, it has
been shown that there are different types of biomolecules inside these
vesicles, including proteins, lipids, and nucleic acids [2]. The lipid
membrane of the EVs encapsulates its contents to protect them from
degradation enzymes in body fluids. This enables EVs to function as an
effective medium of communication between cells. Moving away from
their point of origin, EVs can transmit information to other cells,
thereby modulating the function of the recipient cells [3].
A long non-coding RNA (lncRNA) is a > 200 nucleotide-long
transcript that does not encode proteins [4]. These RNAs were initially
considered “transcriptional noise.” However, with advances in re-
search, it is now known that lncRNAs are evolutionarily conserved,
exist stably in organisms, and have very important functions in reg-
ulating the expression of microRNAs (miRNAs) and epigenetic mod-
ification [5]. Recent studies have confirmed that lncRNAs play an im-
portant role in tumorigenesis and progression; moreover, they
participate in the transcriptional, post-transcriptional, and epigenetic
regulation of some key pathways during tumorigenesis [6,7], such as
cell proliferation, angiogenesis, anti-apoptosis, and tumor invasion [8].
Exosomes encapsulate these functional lncRNAs to play an important
role in the occurrence, development, and drug resistance of tumors.
Therefore, they can be used as biomarkers for the early diagnosis and
prognosis of tumors [9].
Interestingly, there are a variety of methods used in exosomal RNA

Abbreviations: EVs, extracellular vesicles; ABs, apoptotic bodies; ILVs, intraluminal vesicles); MVBs, multivesicular bodies; PM, plasma membrane; TME, tumor
microenvironment; CAFs, cancer-associated fibroblasts; MM, multiple myeloma; MSCs, mesenchymal stem cells; EMT, epithelial-mesenchymal transformation;
ceRNA, competing endogenous RNAs; GBM, glioblastomas; TMZ, temozolomide; GC, gastric cancer; RT-qPCR, reverse transcription quantitative polymerase chain
reaction; dPCR, digital PCR; NGS, Next-generation sequencing; FC, ferrocene
⁎
Corresponding authors.
E-mail addresses: xutianmin@126.com (T. Xu), cuimanhua@126.com (M. Cui).

https://doi.org/10.1016/j.biopha.2019.109716
Received 24 August 2019; Received in revised form 19 November 2019; Accepted 29 November 2019
0753-3322/ © 2019 Department of Gynecology and Obstetrics, the Second Hospital of Jilin University, Changchun, Jilin, China. Published by Elsevier Masson
SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Y. Wu, et al.
research; the most common techniques include RT-PCR, RNA sequen-
cing, and microarray analysis [10]. However, most of the mentioned
methods include a series of complex processes before analysis, such as
exosome isolation, total exosomal RNA extraction, and cDNA synthesis.
For low content exosomes, this tedious process not only reduces the
final RNA content but also increases the possibility of contamination
and inconsistency, thereby making the detection of RNA extremely
difficult [11]. Therefore, an increasing number of studies have focused
on developing simple, sensitive, and accurate research methods.
In this review, we discuss the role of exosomal lncRNAs in tumor-
igenesis and chemoresistance and as potential tumor biomarkers. We
introduce and describe common and newly established techniques used
in exosomal RNA research as well as their advantages and dis-
advantages.
2. Biogenesis and characteristics of exosomes
Exosomes are a class of EVs with a diameter of ∼30–100 nm [3].
During the formation of exosomes, the endosomal membrane buds in-
ward to form the early endosome. Proteins and other biomolecules in
the early endosomes are further packaged into intraluminal vesicles
(ILVs) that aggregate to form large multivesicular bodies (MVBs). These
bodies can be transported to lysosomes for degradation or fuse with the
plasma membrane (PM) to secrete the internal intraluminal vesicles by
exocytosis to form exosomes [12–14] (Fig. 1). Ectosomes and micro-
vesicles are 100–1000 nm [15] in diameter and are usually assembled
at the PM and secreted by its outward budding. ABs have a diameter of
∼50–5000 nm [16]. When apoptotic cells are cleared, the degraded
cellular debris is packaged as ABs and removed from the cell [17].
Exosomes can transmit biological information to recipient cells that
have the characteristics of their own parent cells, thereby functioning in
cell-to-cell communication. Exosomes and recipient cells majorly in-
teract in the following ways: exosomal proteins and nucleic acids are
transferred to the recipient cells by direct fusion with the cell mem-
brane, the recipient cells internalize exosomes by endocytosis, or the
exosomes interact with the recipient cells via receptor-ligand binding
[18].
3. Exosome-derived lncRNAs in the development and progression
of tumors
LncRNAs play an important role in the occurrence and development
of human cancers. Studies have shown that exosomes derived from
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Biomedicine & Pharmacotherapy 123 (2020) 109716
tumor cells can transfer specific lncRNAs to recipient cells that regulate
the tumor microenvironment [19,20] and promote angiogenesis
[21–23], invasion, and migration [24–26] (Fig. 2). Some exosomal
lncRNAs associated with tumor development that have been studied in
recent years are listed in Table 1.
3.1. The role of exosomal lncRNAs in the tumor microenvironment
The occurrence and development of cancer is closely related to the
tumor microenvironment (TME). TMEs are mainly composed of cancer-
associated fibroblasts (CAFs), extracellular matrix, soluble factors, and
some physical factors [36]. Exosomes represent a communication
medium between cells; crosstalk between malignant cells and non-
transformed cells plays an important role in the regulation of tumor
microenvironments.
CAFs are an important part of the tumor microenvironment and can
promote tumor growth, invasion, and metastasis. CAFs in the stroma in
colorectal cancer secrete exosomes rich in lncRNA H19 that reduce the
levels of cleaved caspase-3 and cell apoptosis induced by oxaliplatin
and increase drug resistance in tumor cells. The exosomal lncRNA H19
derived from CAFs decreases the inhibitory effects of miR-141 on Wnt/
β-catenin signaling, thus enhancing the stemness of colorectal cancer
cells. Overexpression of H19 increases the protein levels of β-catenin
and the expression of its downstream genes. However, knocking down
H19 has opposite effects [29].
In multiple myeloma (MM), exosomes secreted by MM cells reduce
the osteogenic ability of mesenchymal stem cells (MSCs). LncRNA
RUNX2-AS1 in MM-exosomes forms an RNA duplex with the osteogenic
transcription factor RUNX2 in MSCs. This hinders the splicing of
RUNX2 and the osteogenic differentiation of MSCs, which is beneficial
for the growth of tumor cells [20].
The interior of solid tumors often contains a hypoxic micro-
environment. To overcome the effect of hypoxia on tumor tissues,
tumor cells secrete exosomes containing lncRNAs to promote their
proliferation and invasion. Under hypoxia, exosomes secreted by the
bladder cancer cell line 5637 contain abundant lncRNA UCA1, which
promotes the epithelial–mesenchymal transformation (EMT) of re-
cipient cells, reshapes the tumor microenvironment, and, subsequently,
promotes tumor growth [28].
3.2. The role of exosomal lncRNAs in angiogenesis
During tumor development, the dynamic balance between the
Fig. 1. Biogenesis of exosomes: Early endosomes are formed by
the invagination of the endosomal membrane. Proteins and nu-
cleic acids in the cell are packaged into ILVs that form MVBs.
Some of the MVBs are transported to the lysosome for degrada-
tion, whereas other MVBs can fuse with the plasma membrane and
the ILVs are secreted from the cell, i.e., as exosomes. Biomolecules
encapsulated in exosomes are transferred to recipient cells by di-
rect fusion, endocytosis, or receptor-ligand binding.
Y. Wu, et al.
angiogenic and anti-angiogenic factors is lost; this leads to a continuous
increase in angiogenic factors and uncontrolled growth of blood vessels
that are beneficial for the growth and invasion of tumors. It has recently
been shown that lncRNAs in the secretions from tumor cells are in-
volved in this process [37,38].
Tumor cell-derived exosomes containing lncRNAs HOTAIR [21],
CCAT2 [22], and POU3F3 [33] in gliomas can increase VEGF, TGFβ,
and FGF expression to promote angiogenesis and enhance Bcl-2 ex-
pression while reducing that of Bax and caspase-3, thus inhibiting hy-
poxia-induced apoptosis. Similarly, in the exosomes secreted by epi-
thelial ovarian cancer cells, the lncRNA MALAT1 promotes
angiogenesis and stimulates the expression of angiogenic genes (in-
cluding VEGF-A, VEGF-D, IL-8, and angiogenin) [23].
3.3. The role of exosomal lncRNAs in tumor invasion and metastasis
Exosomal lncRNAs play an important role in maintaining the
stemness of tumor cells and promoting tumor invasion and metastasis.
LncRNA Sox2ot is transported to adjacent cells and blood by exosomes
secreted by pancreatic ductal adenocarcinoma cells. Upon internaliza-
tion of these exosomes by the recipient cells, the Sox2ot competing
endogenous RNAs (ceRNAs) directly bind miR-200s and upregulate the
expression of Sox2. Subsequently, the expression of E-cadherin (an EMT
marker) decreases and that of N-cadherin and vimentin along with the
proteins involved in cancer stemness, such as CD133, CD44, ALDH1,
and Nanog, increases. These results suggest that the exosomal Sox2ot
promotes EMT and maintains the stemness of tumor cells. When the
pancreatic ductal adenocarcinoma cells treated with Sox2ot-over-
expressing exosomes were implanted into mouse pancreases, their tu-
morigenicity and liver metastasis significantly increased, indicating
that exosomal lncRNA Sox2ot promotes tumor invasion and metastasis
[35]. Similarly, in female esophageal squamous cell carcinoma, the
exosomal lncRNA FMR1-AS1 binds to toll-like receptor 7 activating the
NF-κB signaling cascade and upregulating c-Myc, thereby promoting
the proliferation, invasion, and anti-apoptosis of cancer cells. The
crosstalk between cancer and non-cancer stem cells mediated by FMR1-
AS1 in exosomes maintains the dynamic balance in tumor stemness
[31].
In lung cancer, tumor cell-derived exosomal lncRNA MMP2-2 in-
creases the expression of matrix metalloproteinase 2 and vascular
permeability, which are important for the migration and invasion of
lung cancer cells into blood vessels [25].
4. Exosome-derived lncRNAs and chemoresistance
Increasing drug resistance in cancer chemotherapy (chemoresis-
tance) has severely affected the therapeutic efficacy and survival of
patients with cancer. Many studies have shown that lncRNAs are in-
volved in the regulation of a variety of genes that promote the devel-
opment of cancer chemoresistance. Moreover, it has been observed that
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Biomedicine & Pharmacotherapy 123 (2020) 109716
Fig. 2. The role of exosomes in tumors. Tumor cells can se-
crete exosomes containing functional lncRNA, which transmits
information between cells and tissues, regulates tumor mi-
croenvironments, promotes angiogenesis and invasion, and
increases the drug resistance of tumor cells. At the same time,
it can also be used as a tumor biomarker for diagnosis.
lncRNAs in exosomes increase the resistance of tumor cells to che-
motherapeutic drugs (Table 2).
An increasing number of patients with HER2-positive breast cancer
are resistant to trastuzumab chemotherapy. The expression of lncRNA
APAP2-AS1 is significantly upregulated in drug-resistant cells; this
lncRNA can be packaged into exosomes and released. When the exo-
somes containing lncRNA APAP2-AS1 were incubated with trastu-
zumab-sensitive cells, drug-induced apoptosis was reduced; moreover,
silencing of APAP2-AS1 from the exosomes reversed this drug re-
sistance [39]. Similarly, exosomal lncRNA SNHG14 promotes resistance
to trastuzumab-mediated chemotherapy in patients with HER2-positive
breast cancer [40].
lncRNA SBF2-AS1 is highly expressed in glioblastoma (GBM) cells
resistant to temozolomide (TMZ). SBF2-AS1 functions as a ceRNA for
miR-151a-3p and inhibits the effect of miR-151a-3p on the XRCC4
gene; it reduces the expression of γ-H2AX (a marker of DNA double-
strand break), which increases the repair of damaged DNA, thereby
enhancing the resistance of GBM cells to TMZ. LncSBF2-AS1 can be
packaged into exosomes and transmit chemoresistance between cells
and tissues. After transplanting mice with GBM cells co-incubated with
lncSBF2-AS1-containing exosomes, the tumor grew significantly after
TMZ treatment, whereas using GBM cells co-incubated with SBF2-AS1-
depleted (shSBF2-AS1) exosomes showed a regressed tumor post-TMZ
treatment [43].
Upregulation of serum exosomal lncRNA RP11-838N2.4 induces the
resistance of non-small cell lung cancer cells to erlotinib. Knockdown of
lncRNA RP11-838N2.4 abrogates this effect [45]. Exosomal lncRNA
H19 induces resistance to gefitinib in non-small cell lung cancer [46].
Exosomal lncRNA UCA1 is involved in drug resistance in a variety of
cancers, including resistance to cisplatin in ovarian cancer [47], ce-
tuximab in colorectal cancer [44], and tamoxifen in breast cancer [41].
These studies prove that exosomes transport lncRNAs to induce drug
resistance in tumor cells and transfer chemoresistance between cells
such that sensitive cells become chemoresistant. Exosomes carrying
lncRNAs associated with drug resistance often predict poor outcomes in
tumor patients. Therefore, exosomes have the potential to be used as
novel prognostic indicators and therapeutic targets for drug resistance.
5. Exosome-derived lncRNAs as tumor biomarkers
Exosomes derived from tumor cells have the same biomolecular
characteristics as their parent cells. The lncRNAs are encapsulated by
the exosomal membrane and exist stably in various parts of the human
body. Functioning as new non-invasive tumor diagnostic biomarkers
(Table 3), exosomal lncRNAs have gained an increasing amount of at-
tention and have been found in a variety of cancers. Many studies have
found that the levels of lncRNAs present in exosomes are often asso-
ciated with the stage of the tumor, its recurrence, and the survival of
patients; thus, they can be used to evaluate the prognosis of tumors.
Upon analyzing multiple logistic regression models and receiver
Y. Wu, et al. Biomedicine & Pharmacotherapy 123 (2020) 109716

Table 2

[27]
[28]

[24]
[29]
[23]
[30]
[31]

[32]

[21]
[22]

[33]
[34]
[25]

[20]

[26]

[35]
Refs The role of exosomal lncRNAs in drug resistance in tumors.

Inducing proliferation, anti-apoptosis, and invasion of esophageal squamous cell carcinoma cells

Destroying the ability of osteogenic differentiation of MSCs and promoting the growth of tumor

Promoting the growth and migration of tumor cells and preventing the apoptosis of tumor cells
Cancer type LncRNA Resistant drugs Refs
Reshaping the tumor microenvironment to promote cell proliferation, migration, and invasion

Regulating vascular permeability and migration and invasion of lung cancer cells into blood
Breast cancer AGAP2-AS1 trastuzumab [39]

and maintaining a dynamic balance between cancer stem cells and non-cancer stem cells
SNHG14 trastuzumab [40]

Maintaining the stemness of tumor cells and promoting tumor invasion and metastasis
UCA1 tamoxifen [41]
Esophageal squamous cell Part1 gefitinib [42]
carcinoma
Glioblastoma lncRNA SBF2- temozolomide [43]
AS1
Maintaining the stemness of tumor cells and promoting drug resistance

Metastatic colorectal cancer UCA1 cetuximab [44]

Non-small cell lung cancer RP11-838N2.4 erlotinib [45]
H19 gefitinib [46]
Ovarian cancer UCA1 cisplatin [47]

Promoting cell proliferation, migration, and angiogenesis

Renal Cancer ARSR Sunitinib [48]

operating characteristic curves, it was observed that lncRNAs pCAT-1,

Promoting tumor cell migration and invasion

Promoting tumor cell migration and invasion

UbC1, and SNHG16 in the serum of patients with bladder cancer pro-
Promoting cell proliferation and migration

vided a better diagnosis with significantly higher accuracy compared to

the use of urine cytology. Multivariate COX analysis showed that UBC1
is independently associated with tumor recurrence in non-muscular
invasive bladder cancer [49]. Thus, it can be used as a biomarker for the
Promoting cell proliferation

Promoting cell proliferation

prognosis of bladder cancer. Similarly, urine-derived exosomal lncRNAs

Promoting angiogenesis

Promoting angiogenesis
Promoting angiogenesis

MALAT1, PCAT-1, and SPRY4-IT1 are used in diagnosing and evalu-

ating the prognosis of bladder cancer [51].
Circulating exosomal miRNA-21 and lncRNA-ATB are important for
TNM staging and the prognosis of hepatocellular carcinoma.
Function

Multivariate analysis of Cox regression tests showed that the levels of

vessels

cells

exosomal miRNA-21 and lncRNA-ATB correlate positively with tumor

size; increased expression of these non-coding RNAs (ncRNAs) was an
Enhancing metastasis of colorectal cancer by modifying HNRNPK expression

Activating VEGFA and transforming growth factor β promoted the expression

Activating the TLR7-NFκB signaling pathway and upregulating c-Myc levels

Increasing gene and protein expression of bFGF, bFGFR, VEGFA, and Angio

independent predictor of mortality and disease progression. The overall

Knocking out ZFAS1 inhibits the cell cycle, induces apoptosis, and inhibits
Promoting tumor growth and progression through epithelial-mesenchymal

Competitive binding of miR-200 family to regulate the expression of Sox2

Promoting cell cycle, accelerating tumor proliferation and metastasis, and
Inhibiting RUNX2 (the main regulator of bone formation) and osteogenic

survival and progression-free survival rates are significantly lowered in

patients with a high expression of circulating miRNA-21 and lncRNA-
ATB. This finding shows that circulating exosomal miRNA-21 and
Promoting the expression of matrix metalloproteinase-2 (MMP2)
Competing with miR-141 and activating the β-catenin pathway

lncRNA-ATB can be used as ideal non-invasive serum tumor markers to

of Bcl-2 and inhibited the expression of Bax and caspase-3
Binding to miR-326 to promote the growth of tumor cells

predict the prognosis of liver cancer [62].

Stimulating the expression of angiogenic related genes

Serum lncRNA-containing exosomes also play a very important role

in the diagnosis and prognosis of gastric cancer (GC). It was shown that
differentiation of mesenchymal stem cells (MSC)

the expression of serum exosomal HotTip in patients with GC positively

correlates with the depth of tumor invasion and clinical stage; HotTip
epithelial-mesenchymal transformation

negatively correlates with overall survival rate, which is an in-

Inhibiting miR-204-3p in astrocytes

dependent prognostic factor in patients with GC. Moreover, it is a better

indicator for diagnosis than the commonly used tumor markers CEA,
inhibiting tumor cell apoptosis

CA19-9, and CA72-4 [59]. The study also found that the lncRNA UEGC1
in the exosomes secreted by cancer cells can be stably present in the
plasma of patients with early gastric cancer. The diagnostic accuracy
provided by UEGC1 is higher than that provided by CEA [58].
in recipient cells

In conclusion, tumor cells can secrete exosomes containing specific

Mechanism

transition

functional lncRNAs that transmit information between cells and tissues,

thus promoting tumor progression. Once these exosomes are inter-
The role of exosomal lncRNAs in tumor progression.

nalized by the recipient cells, the lncRNA(s) regulate a specific sig-

naling pathway in the form of ceRNAs [35,43] or form double-stranded
RUNX2-AS1
FMR1-AS1

structures with the target gene(s) to inhibit its expression [20]. Exo-
MALAT1

MALAT1

MMP2-2
HOTAIR

POU3F3
LncRNA

Malat-1
CCAT2
PCAT1

Sox2ot
ZFAS1

somal lncRNAs can transform the epithelial phenotype of tumor cells to

UCA1

ATB
91H
H19

a mesenchymal phenotype, i.e., positively regulate the EMT of tumor

cells [28,32], loosen the structure of the surrounding extracellular
matrix, and increase the permeability of blood vessels [25], thereby
Pancreatic ductal adenocarcinoma

enabling the invasion and metastasis of the tumor cells. These exosomes
can also regulate adjacent non-tumor cells by promoting the secretion
Non-small cell lung cancer
Esophageal squamous cell
Epithelial ovarian cancer

of angiogenic factors by endothelial cells and reducing endothelial cell

apoptosis in anoxic environments, thereby enhancing the resistance of
Multiple myeloma
Colorectal Cancer

tumors to hypoxia [21], which is then conducive to tumor growth.

Bladder cancer

carcinoma

Gastric cancer
Breast cancer
Cancer Type

Exosomal lncRNAs can also increase the repair of damaged DNA, pro-
Lung cancer

vide resistance to chemotherapeutic drugs, and transmit this che-

Gliomas
Table 1

moresistance to sensitive cells. Exosomal lncRNAs have multifaced roles

in tumors. In recent years, with the advancements in research,

4
Y. Wu, et al. Biomedicine & Pharmacotherapy 123 (2020) 109716

Table 3
The role of exosomal lncRNAs in tumor diagnosis and prognosis.
Cancer type LncRNA Gene expression Sample source Refs

Bladder cancer pCAT-1, UbC H19 UP Serum [49,50]

MALAT1, PCAT-1, SPRY4-IT1 UP Urine [51]
Breast cancer HOTAIR UP Plasma [52]
Cervical cancer HOTAIR, MALAT1 Up Cervicovaginal lavage [53]
MEG3 Down
Colorectal cancer LNCV6_116109, LNCV6_98390, LNCV6_38772, LNCV_108266, LNCV6_84003, UP Plasma [54]
LNCV6_98602
CRNDE-h Up Serum [55,56]
CCAT2
Epithelial ovarian cancer MALAT1 UP Serum [57]
Gastric cancer UEGC1 UP Plasma [58]
HotTip UP Serum [59]
Glioblastoma multiforme HOTAIR UP Serum [60]
Hepatocellular carcinoma HEIH lncRNA-ATB ENSG00000258332.1, LINC0063 LINC00161 UP Serum [61–64]
Lung squamous cell carcinoma SOX2-OT UP Plasma [65]
Non‐small‐cell lung cancer Gas5 Down Serum [66]
Prostate cancer P21 UP Urine [67]
SAP30L-AS1, SChLAP1 UP Plasma [68]

exosomes represent novel candidates for use in the study of the me-
chanism(s) of tumorigenesis and targeted therapy. As exosomes can
exist widely and stably in body fluids and the lncRNAs present have
similar biological characteristics as the parent tumor cells, they have
gained acceptance as a new type of tumor marker for the diagnosis and
prognosis of cancers.
6. Methods used for exosomal ncRNAs
In addition to lncRNAs, other ncRNAs, such as miRNAs and
circRNAs, in exosomes also play important roles in tumorigenesis
[69–71]. There are some limitations to the methods used to separate
exosomes, such as poor purity and low yield. Isolated exosomes often
contain ribonucleoprotein complexes, lipoproteins, and other con-
taminating molecules. Low levels of RNA can easily amplify the impact
of biological contamination in the samples, which leads to inaccurate
analysis of exosomal RNA [72,73]. Existing separation techniques
mainly include centrifugation, filtration, immunoaffinity isolation, and
polymeric precipitation [74]. Different separation methods have dif-
ferent effects on downstream analysis. The purity of exosomes obtained
by differential centrifugation and density gradient centrifugation is
higher, with lower yields, than that obtained by the other techniques.
Filtration uses a physical barrier to separate and obtain exosomes based
on the size of the vesicles; however, passing these vesicles through the
filter hole may lead to a change in their shape or rupture, thereby af-
fecting the downstream analysis [75]. The specificity of immunoaffinity
isolation is high, but labeling is not equal on all types of vesicles.
Polymeric precipitation is simple and fast, but the purity obtained is
poor. The impurities in polymeric precipitation increase the content of
RNA, making it easy to obtain high RNA yields [76]. Methods used for
separation are primarily chosen based on the research problem(s) being
addressed. For example, to determine whether exosomal RNA is asso-
ciated with a disease or to carry out functional research, it is necessary
to choose a separation method with high purity or else the con-
taminants carried over during the separation process may give rise to
inaccuracies. However, a technique involving lower purity can be se-
lected when exosomal RNA is used as a biomarker for diagnosis, its
relationship with the disease is clear, the test results are reproducible,
and the success rate of diagnosis is high [77].
Once the exosomal RNA is isolated from the sample, it needs to be
analyzed. Existing techniques in exosomal ncRNA research mainly in-
clude quantitative PCR (qPCR) [78–80], RNA sequencing [81–83], and
microarrays [84–86]. These are novel detection methods that have
arisen from the urgent need for simple, easy-to-operate, highly sensi-
tive, and highly accurate exosomal ncRNA research methods.
6.1. Methods based on PCR
Reverse polymerase chain reaction (RT-qPCR) is one of the main
methods used to study exosomal ncRNAs. Using the basic principles of
PCR, RT-qPCR quantitatively detects the target gene by monitoring
amplified fluorescence signals in real-time. However, this is a com-
parative quantitative method that quantifies the target genes with the
help of a standard curve generated from a known copy number of the
reference gene(s) [87]. In this technique, the amplification efficiency of
samples and standards needs to be consistent [88]. RT-qPCRb tech-
nology is widely used in the study of exosomal ncRNA in a variety of
cancers, such as breast cancer [52], lung cancer [66], prostate cancer
[68], and multiple myeloma [89]; these lncRNAs are related to the
development of tumors [27,28]. Through RT-qPCR detection, re-
searchers found that exosomal lncMALAT1 is highly expressed in pa-
tients with epithelial ovarian cancer and is associated with its prog-
nosis. The content of exosomal lncMALAT1 in the serum, the tumor
FIGO stage, and lymph node metastasis can accurately predict the
three-year overall survival of patients with ovarian cancer [23]. Si-
multaneously, RT-qPCR confirmed that the expression of exosomal
lncSBF2-AS1 in the serum of patients with recurrent glioblastoma was
significantly higher than that in the serum of primary patients, which
suggested that tumor cells were not sensitive to temozolomide and the
prognosis of patients was poor [43].
6.1.1. Advantages
This technique is methodical and widely used in RNA research. It
can be automated with the help of a machine to quantify RNA with high
sensitivity.
6.1.2. Disadvantages
Only target genes corresponding to the designed primers can be
studied, and the technique cannot be used for the detection of total RNA
and unknown gene fragments [11]. The diversity in the detection re-
agents and reaction cycle systems and the lack of homogeneity in the
selection of reference genes and data analysis make it difficult to
standardize the detection of exosomal ncRNAs with RT-qPCR [90].
Moreover, low abundance or low specificity gene sequences cannot be
detected. Therefore, researchers have turned their attention to a more
accurate PCR technique, i.e., digital PCR (dPCR). With the advances in
microfluidics, PCR has evolved from being qualitative to semi-quanti-
tative and, finally, to absolute quantitative dPCR.
Principles of dPCR (Fig. 3): nucleic acid-containing samples are
divided into many chambers using specific sample dispersion methods,
including those based on droplets, channels, and hydrogel beads. Each

5
Y. Wu, et al.
chamber is a separate compartment that usually contains zero or one
molecule. Few small chambers contain two or more molecules; these
are excluded by identifying their abnormal fluorescence signals [91].
Each chamber is used as a separate reaction system to amplify the
nucleic acid sequence of interest. A chamber containing the target gene
is detected using a probe for a positive fluorescence signal, and the
chamber with a positive signal is counted. The target gene is quantified
by calculating the proportion of positive chambers to the total [92,93].
Depending on the different methods of sample dispersion, there are two
main analysis protocols for quantifying fluorescence signals. One pro-
tocol involves the counting of fluorescence signals based on droplets,
similar to the principles of flow cytometry. The droplets in each com-
partment pass through the detector, and each droplet obtains a fluor-
escence value; the proportion of the number of positive droplets (in-
cluding target genes) to the number of total droplets is the absolute
value of the target gene present [94]. Another method is based on chip-
based fluorescent point image processing using a two-dimensional
fluorescence bitmap to quantify the target gene. Bai et al. developed a
new multi-color fluorescence digital PCR EV-lncRNA analysis chip,
which can quickly and accurately distinguish between patients with
lung cancer and healthy individuals by detecting the expression levels
of exosomal lncSLC9A3-AS1 and lncPCAT6. This method is more sen-
sitive than qRT-PCR [95].
6.1.3. Advantages
This method does not require reference genes or standard curves
and is not affected by the efficiency of amplification. It is an absolute
quantitative method. It possesses high sensitivity and precision and can
be used to quantify samples with low abundance and rare sequences. It
has been used to analyze heterogeneities in tumors, liquid biopsies, and
other research areas related to cancer [93]. Studies have compared
qPCR with dPCR in their practical application. The results show that,
although dPCR and qPCR are consistent in their results, the former has
higher precision, a lower detection limit, reproducibility, and stronger
tolerance to inhibitors [95–97].
6.1.4. Disadvantages
dPCR is a low-throughput method that requires concentrated sam-
ples to be diluted. The cost of operation is high, and there are very few
laboratories with the corresponding testing equipment. The application
of dPCR in experimental and clinical detection is also limited.
6.2. Methods based on microarrays (biochip)
Microarrays or biochips are a high-throughput gene detection
method based on molecular hybridization. A modified DNA probe is
fixed onto a solid surface and produces a fluorescence signal when it
binds to a specific target gene in the sample. The expression of each
sequence is determined by analyzing the fluorescence signal.
The exosomal RNA obtained by conventional methods is derived
from all the exosomes in the body; of these, some are released by tumor
cells and others by non-tumor cells. Therefore, the sensitivity and
specificity of tumor-associated exosomal RNA analysis are often re-
duced. To solve this problem, Yang et al. developed an immune biochip
6
Biomedicine & Pharmacotherapy 123 (2020) 109716
Fig. 3. Principles of dPCR. The gene fragments in the sample
are scattered into many chambers; each compartment contains
zero or one gene fragment. As each chamber is an independent
compartment, the gene fragments can be amplified separately,
and the target gene can be detected by probing. The positive
fluorescence signal is counted, and the target gene can be
quantified by calculating the ratio of the positive fluorescence
signal to the total signal.
that specifically enriches exosomes containing the EGFR or PD-L1
tumor biomarkers from the serum of patients with lung cancer. This
separates the tumor-derived exosomes from the total exosomes in the
serum. Cationic lipoplexes containing RNA target-sensing molecular
beacons are used to detect the expression of miR-21 and TTF-1 mRNA
in the tumor-derived exosomes in situ. Fluorescence signals are gener-
ated upon combining the molecular beacons with the target gene, and
the amount of the target exosomal RNA can be quantified by measuring
this fluorescence intensity. This method can distinguish between the
tumor-derived exosomes and non-tumor-derived exosomes, thereby
reducing the noise by non-tumor-derived exosomal RNA and increasing
the sensitivity and specificity compared to whole serum detection. This
method is more simple and effective than the common workflow in-
volved in immunomagnetic separation-RNA isolation-RT-qPCR [98].
6.2.1. Advantages
This method is simple, rapid, and requires less sample volume;
moreover, it is widely used in the detection of exosomal RNA.
6.2.2. Disadvantages
This method is expensive and prone to contamination by DNA.
When the probe is hybridized with the sample gene, a false positive
signal can easily be obtained. Its specificity needs to be improved. This
method can only detect known RNA sequences [99].
6.3. Methods based on next-generation sequencing (NGS)
Next-generation sequencing is a high-throughput gene detection
method that identifies new mutations while determining the char-
acteristics of the disease [100]. This method has been increasingly used
in RNA research owing to the reductions in its cost. Jin et al. analyzed
plasma exosomal miRNA in 41 patients with non-small-cell lung cancer
and 42 healthy individuals by the NGS technique and verified the dif-
ferentially expressed genes. The results showed that miR-181-5p, miR-
30a-3p, miR-30e-3p, and miR-361-5p in exosomes were adenocarci-
noma-specific, whereas miR-10b-5p, miR-15b-5p, and miR-320b were
squamous cell carcinoma-specific [101]. The same method was used in
the study of diagnostic markers for papillary thyroid cancer and nod-
ular goiter [102] as well as for the occurrence and development of
colorectal cancer [29].
6.3.1. Advantages
NGS can detect all the miRNAs and lncRNAs in exosomes and can be
used to study the differential expression of each gene [81–83]. It can be
used to detect the purity of exosomes by detecting foreign genetic
material (such as interference from fetal bovine serum) [11].
6.3.2. Disadvantages
The cost of NGS is high. This method is time-consuming, involves a
large volume of data during analysis, and the results need to be verified.
Therefore, this method cannot be used for large-scale clinical diagnosis.
Y. Wu, et al.
6.4. Methods based on fluorescing molecular beacons
Detecting fluorescence from molecular beacons has been used to
detect exosomal RNA in breast [103], prostate [104,105] pancreatic
[106], and lung cancers [107]. Various exosome isolation methods may
lead to RNA damage that may affect the study [108]. Therefore, Zhai
et al. synthesized a nucleic acid-functionalized gold nanoparticle probe
that can directly enter exosomes present in the plasma that specifically
bind to miR-1246 and generate fluorescence. The differences in gene
expression between the two groups were compared by analyzing the
fluorescence signals used in diagnosing breast cancer.
6.4.1. Advantages
This method does not require the separation of exosomes from the
plasma or the extraction of RNA from the exosomes. Being a non-in-
vasive diagnostic method for breast cancer, its advantages include
simple operation, quick execution, high sensitivity and specificity, and
the requirement of a small sample volume [103].
6.4.2. Disadvantages
The fluorescence background limits the concentration of the probe.
Increasing the concentration of the probe can increase the speed of its
entry into exosomes but also increase the background fluorescence. The
penetration ability of the gold nanoparticle probe depends on its size.
Therefore, the size of the gold nanoparticles determines the time and
ratio of the entry of the probe into the exosomes. At the same time, the
method needs to be verified in more patients.
6.5. Methods based on electrochemical sensors
Electrochemical sensors are used for exosomal RNA detection and
are very sensitive, specific, and reproducible [109,110]. Zhang et al.
designed an electrochemical sensor based on a bipedal DNA walker (a
DNA walker consists of two catalyst domains as its double “legs” and a
linear domain as the “body”) that can detect breast cancer cell and
serum-derived exosomal miR-21. The capture probe labeled with a
locked nucleic acid is fixed on the surface of magnetic beads, and the
DNA walker is combined by hybridizing with a probe. When exosomal
miR-21 enters the system, miR-21 specifically combines with the cap-
ture probe using a toehold-mediated strand displacement reaction to
replace the DNA walker so that it can be released into the solution. The
probe with magnetic beads is pulled by the magnet towards the bottom
of the reaction tube, and the released DNA walker lies in the super-
natant. Subsequently, a drop of the supernatant is placed on the elec-
trode surface, and the DNA walker binds to the methylene blue-mod-
ified hairpin probe 1 on the electrode surface. A ferrocene (FC)-labeled
hairpin probe 2 is added; this replaces the DNA walker combined with
probe 1 to move the DNA walker to the next probe. After multiple cy-
cles, increasing amounts of FC is adsorbed on the electrode surface.
Exosomal miR-21 can be quantified by measuring the ratio of the
electrical signals produced by FC and methylene blue [110].
6.5.1. Advantages
As the DNA walker can continuously amplify the electrical signal
during its movement, the sensor possesses very high sensitivity.
Moreover, the locked nucleic acid and toehold-mediated strand dis-
placement reaction make the sensor very specific, such that even a
single base-mismatched target can be filtered out. The sensor is capable
of regenerating quickly (i.e., even after five repeated activations, the
sensor maintains its stability and sensitivity).
6.5.2. Disadvantages
The preparation process of the sensor is complex, and the sample
needs to be transferred from the reaction tube equipped with probes to
the electrode surface, which could lead to loss of some of the sample.
Additionally, this method cannot be widely used in the detection of
7
Biomedicine & Pharmacotherapy 123 (2020) 109716
exosomal ncRNA.
7. Conclusion
Exosomes are extracellular vesicles that carry biomolecules, in-
cluding proteins, RNA, DNA, and lipids, from parent cells and transfer
them to recipient cells, thereby regulating the biological behavior of the
recipient cells. An increasing number of studies have found that exo-
somes are carriers of biological information and play an important role
in tumor progression. Tumor-derived exosomal lncRNAs promote an-
giogenesis, inhibit immune cell function, and regulate the tumor mi-
croenvironment; all the processes are conducive to the migration of
tumor cells [30,32,34,111]. Exosomal lncRNAs have also been used in
the early diagnosis, evaluation of the prognosis, and treatment of cancer
[112,113]. However, exosomes are heterogeneous, and those derived
from tumors have multiple subtypes with different functions [114,115].
Moreover, there is limited knowledge available regarding the subtypes
of exosomes and the mechanism(s) involved in the loading of RNA into
exosomes [116–118]. Understanding the subtypes of exosomes and the
loading mechanism of RNA is important; thus, these aspects need to be
addressed in the future. This is imperative for classifying these subtypes
and identifying their functions; it will also help to elucidate the me-
chanism underlying the regulation of exosomal RNA in the pathophy-
siological processes associated with tumors and lay the foundation for
exosome-targeted tumor therapy [119].
Studying exosomal RNA is extremely challenging. The existing se-
paration and purification methods have some disadvantages [9,120]
that result in low exosomal yield or low purity, thereby making the
process extremely difficult to understand. Researchers have established
techniques for collecting, separating, and purifying exosomes from
various samples [121] and have provided detailed experimental ana-
lyses [11,122,123] that can improve the accuracy of the results to a
certain extent. However, a technique that produces high yields and pure
exosomes remains to be developed. Using the current array of techni-
ques, the most appropriate separation method can be selected ac-
cording to the question being addressed and the downstream target(s).
Currently, the methods used in exosomal RNA research mainly in-
clude RT-PCR, NGS, and microarrays. However, these methods have
some limitations. In recent years, with advances in detection techniques
and instruments, some new detection methods, such as dPCR, have also
been used in the study of exosomal RNA. Techniques that are sensitive,
specific, simple, and quick, such as gene chips, molecular beacons, and
electrochemical sensors, have recently emerged. However, these are not
commonly used. Therefore, we urgently need to identify new methods
for the identification, classification, and high-throughput analysis of
exosomes to better understand their roles in disease.
Declaration of Competing Interest
The authors declare they have no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (grant numbers 81772772 and 81302242); the
Jilin Province Science and Technology Funds (grant number
20170622008JC).
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