Biomedicine & Pharmacotherapy 82 (2016) 509–519

Available online at

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Differential role of microRNAs in the pathogenesis and treatment of

Esophageal cancer
Maryam Hemmatzadeha,b,c,1, Hamed Mohammadia,c,1, Mohammad Karimia,b,c,
Mohammad Hossein Musavishenasa,b,c , Behzad Baradarana,*
a
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
b
Tabriz University of Medical Sciences, International Branch (Aras), Tabriz, Iran
c
Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

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

Article history:
Received 20 February 2016 Esophageal cancer (EC) is the most invasive disease associated with inclusive poor prognosis. EC usually
Received in revised form 6 May 2016 is found as either adenocarcinoma (EAC) or squamous cell carcinomas (ESCC). ESCC forms in squamous
Accepted 9 May 2016 cells and highly occurs in the upper third of the esophagus. EAC appears in glandular cells and ordinarily
develops in the lower one third of the esophagus near the stomach. Barrett’s esophagus (BE) is a
Keywords: metaplastic precursor of EAC. There is a persistent need for improving our understanding of the
MicroRNA molecular basis of this disease. MicroRNAs (miRNAs) demonstrate an uncovered class of small, non-
Esophageal cancer coding RNAs that can negatively regulate the protein coding gene, and are associated with approximately
Treatment
all known physiological and pathological processes, especially cancer. MiRNAs can affect cancer
pathogenesis, playing a crucial role as either oncogenes or tumor suppressors. The recent emergence of
observations on the role of miRNAs in cancer and their functions has induced many investigations to
examine their relevance to esophageal cancer. In esophageal cancer, miRNA dysregulation plays a crucial
role in cancer prognosis and in patients’ responsiveness to neo-adjuvant and adjuvant therapies. In this
review, the oncogenic, tumor suppressive, and drug resistance related roles of miRNAs, and their
involvement in the pathogenesis and treatment of esophageal cancer were summarized.
ã 2016 Elsevier Masson SAS. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
2. Esophageal cancer subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
2.1. Esophageal adenocarcinoma (EAC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
2.2. Esophageal squamous cell carcinomas (ESCC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3. MiRNAs in the pathogenesis of EC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3.1. Dysregulation of miRNAs in EAC and its precursor BE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
3.2. Dysregulation of miRNAs in ESCC development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
4. MiRNAs as biomarkers in esophageal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
5. Oncogenic miRNAs in esophageal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
5.1. MiR-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
5.2. MiR-10b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
5.3. MiR-17-92 polycistron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
5.4. MiR-200c and miR-221/222 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
6. Tumor suppressor miRNAs in esophageal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
6.1. Let-7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
6.2. MiR-375 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

* Corresponding author.
E-mail address: Behzad_im@yahoo.com (B. Baradaran).
1
These authors contributed equally to this manuscript.

http://dx.doi.org/10.1016/j.biopha.2016.05.009
0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
510 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519

6.3. MiR-145 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

6.4. MiR-34a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
7. Chemoresistance miRNAs in esophageal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
7.1. MiR-148a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
7.2. MiR-141 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
7.3. MiR-27a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
7.4. MiR-296 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
8. Potential in screening and treatment of EC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
9. MiRNAs as therapeutic targets in esophageal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517

1. Introduction 2. Esophageal cancer subtypes

Esophageal cancer is the eighth most occurring, and the sixth 2.1. Esophageal adenocarcinoma (EAC)
most fatal cancer worldwide [1]. Histologically, in almost 95% of
cases, the disease is found as ESCC or EAC, with the latter being Esophageal adenocarcinoma (EAC), ordinarily forms in the
approximately 50% more prevalent in the United States but at least lower one third of the esophagus and arises from the Barrett’s
20-times less common in Asian countries [1,2]. The EC occurrence esophagus (BE) [7]. EAC, considered as a consequence of chronic
in the high-risk northern Chinese is more than 100/100000, gastroesophageal reflux, is preceded by the progression of BE, a
whereas in the low-risk western Africa can be 20-fold lower [3]. premalignant condition comprising intestinal metaplasia that
ESCC remains the main subtype of EC, especially in China; in progresses as a sequel of gastroesophageal reflux. Not all BE
contrast, EAC is the most common type in Western countries. The patients progress EAC; nevertheless, in a patient with BE, the risk of
pathogenesis of EC remains unclear. 14–21% of cancers (T1 lesions) EAC is 30 times higher than in the general population [15]. Other
and 38–60% of cancers that invade muscle (T2 lesions) are markers of gastroesophageal reflux disease, such as esophageal
associated with spread to lymph nodes [4,5]. ulcer and frequent utilization of the histamine-H2 blockers, are
The most prevalent risk factor for EAC is chronic gastroesopha- also related to the increased risk but do not seem to be the
geal reflux disease, which triggers inflammation in the distal independent risk factors [16]. Drugs that relax the gastroesopha-
esophagus, resulting in progression of the premalignant lesion geal sphincter and augment the reflux, such as anticholinergic
known as BE or intestinal metaplasia [6]. The development of BE to agents, aminophyllines, and beta-blockers, may contribute to the
EAC, progresses through the established histological changes: development of up to 10 percent of these cancers [17].
intestinal metaplasia (BE) to low-grade dysplasia (LGD) to high
grade dysplasia (HGD) to EAC [7]. There is an insistent need for 2.2. Esophageal squamous cell carcinomas (ESCC)
better understanding of the molecular characteristics of the
disease for the progression of clinically useful biomarkers and Esophageal squamous cell carcinoma arises from the squamous
therapeutic modalities. Biomarkers have been explored based on epithelium of the esophagus and is associated with tobacco
the modifications in genomic DNA, and in the expression of specific consumption [7]. Any factor that causes the chronic irritation and
mRNA or protein molecules, or the metabolites, for many years inflammation of the esophageal mucosa seems to increase the
now [8]. MiRNAs have emerged as a novel class of biomolecules prevalence of squamous-cell carcinoma of the esophagus. Sub-
with significant roles in cellular functions in both healthy and stantial alcohol intake, especially in cooperation with smoking,
diseased cells, and have distinguished potential as biomarkers. greatly increases the risk of squamous-cell carcinoma (but not
Functionally, active miRNAs or mature miRNAs, are 18–22 adenocarcinoma) [18]. In Asia, 90% of EC has the histological type of
nucleotide long, single- stranded RNA molecules with 50 phosphate ESCC. Currently, the most operative treatment is surgical resection.
and 30 hydroxyl groups [2]. Pre-miRNAs are created in the nucleus The 5-year survival rate is 20–30 percent for patients who have
by the act of RNAse III endoribonuclease, Drosha. Another curative surgery and do not progress lymph node metastases;
cytoplasmic RNAse III enzyme, Dicer, is bearing stems from the nevertheless, the survival rate is only 13% for patients with at least
stem-loop structures of pre-miRNAs [9]. The double-stranded 1 lymph node metastasis [19].
RNA-binding proteins DGCR8 (DiGeorge critical region 8) and TRBP
(Trans activating response RNA binding protein), work along with 3. MiRNAs in the pathogenesis of EC
Drosha and Dicer, respectively [2]. The pre-miRNA with a typical 50
phosphate and 2-nucleotide 30 overhang is exported into the 3.1. Dysregulation of miRNAs in EAC and its precursor BE
cytoplasm, through exportin 5 (Exp-5), a Ran-dependent nuclear
transport receptor protein [10]. Mature miRNAs motivate their Most EAC cases are caused by BE, a precursor lesion in which the
function by the multi-protein RNA-induced silencing complex squamous epithelium of the esophagus is substituted by a
(RISC) that is also responsible for the phenomenon of RNA metaplastic columnar epithelium. BE is approximated to be
interference, generated by small interfering RNAs (siRNAs). present in 1–2 percent of the general population and confers a
MiRNAs are loaded on RISC complexes where they are unfold into 30-fold increased risk of progressing EAC. The malignant
two single-stranded, mature miRNAs [11,12]. Consequently, a development of BE normally follows the sequence of metaplasia,
mature miRNA can target hundreds of various mRNAs, and the low-grade dysplasia (LGD), high-grade dysplasia (HGD), and
same mRNA can be targeted by the majority of various miRNAs adenocarcinoma [20].
[13,14] (Fig. 1). In this review, we discussed about the role of Some studies have demonstrated the role of miRNAs in the
various miRNAs and their role in pathogenesis and treatment of disease development from Barrett’s to adenocarcinoma. For
esophageal cancer (Table 1). example, Yang et al. [21] compared the samples from BE patients
 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519 511

Fig. 1. Biogenesis of microRNAs. MicroRNAs are transcribed by RNA polymerase II to long primary-miRNA that can be recognized and cleaved by Drosha in the nucleus. These
miRNA is then processed into 70–120-nucleotide-long precursor RNA (pre-miRNA). By exportin 5, a Ran-dependent nuclear transport receptor protein, the pre-miRNA is
exported from the nucleus to the cytoplasm is further processed by Dicer, to produce a duplex microRNA. The passenger miRNA fragment is degraded, whereas the mature
miRNA molecule binds to an Ago protein and incorporates into the RISC. Finally, mature miRNA causes translational repression and target mRNA degradation. DGCR8:
DiGeorge syndrome critical region gene 8, TRBP: Tandem repeat binding protein, RISC: RNA-induced silencing complex.

with low-grade dysplasia, high-grade dysplasia and EAC. They
were incapable to discover any remarkable differentially expressed
miRNAs between BE with LGD and the paired normal tissues.
When they examined HGD and EAC tissues, they found 24 miRNAs
that revealed the modified expression compared to the normal
tissue (14 up-regulated in the diseased tissues and 10 down-
regulated in the diseased tissues). Nine of the 14 up-regulated
miRNAs (hsa-miR-126, hsa-miR-143, hsa-miR-145, hsa-miR-181a,
hsa-miR-181b, hsa-miR-199a, hsa-miR-28, and hsa-miR-30a-5p)
demonstrated greater expression in EAC compared to HGD; 7 of the
10 down-regulated miRNAs (hsa-miR-149, hsa-miR-203, hsa-miR-
210, hsa-miR-27b, hsa-miR-513, hsa-miR-617, and hsa-miR-99a)
revealed lower expression in EAC compared to HGD. These data
seem to offer that miRNA could play a critical role in the
development of the disease to later stages.
Feber et al. [22] compared the expression of 328 human miRNAs
in 10 EAC, 10 ESCC, 5 BE, 1HGD, and 9 normal squamous epithelium
(NSE) tissues. These studies showed that the miRNA expression
profiles of BE and EAC were similar. The single HGD specimen had a
miRNA expression profile similar to EAC samples. About 30
miRNAs were remarkably altered in EAC tissues compared to NSE,
like upregulation of miR-21, miR-192, mir-194 and miR-93 and
down-regulation of miR-203, miR-205, miR-27b, miR-100, miR-
125b, and let-7c. Additionally, it seemed that miR-21, miR-194, and
miR-192 were progressively upregulated from NSE to BE and to
EAC, offering that miRNA dysfunction is an early incident during
512 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519

Table 1 ESCC cancers. Ogawa et al. [27] quantified the expression of 73

Oncogenic oncosuppressor and Chemoresistance function of MicroRNA in
miRNAs through quantitative reverse transcriptase polymerase
Esophageal cancer.
chain reaction (qRT-PCR) in 30 ESCC tumor and paired normal
Name Tumor Type Target References tissues. About 21 miRNAs, including miR-21, miR-25, and miR-151,
Oncogenic microRNAs were upregulated at least 2-fold; and 4 miRNAs were down-
miR-196a EAC ANXA1, SPRR2C, S100A, KRT5 [35,82] regulated at least 2-fold (miR-133a, miR-133b, miR-139, and miR-
miR-25 EAC Bim, CDH1 [22]
145) in ESCC. Hong et al. [24] determined a panel of 12 miRNAs,
miR-93 EAC P21 [23]
miR-221/222 EAC P27kip1, CDX2 [55]
variously expressed between ESCC and normal tissues with 9
miR-21 ESCC PDCD4 [45] upregulated (miR-155, miR-100, miR-146, miR-296, miR-10b, miR-
miR-10b ESCC KLF4 [50] 203, miR-483, miR-494 and miR-220) and 3 down-regulated (miR-
miR-31 ESCC PPP2R2A, PDCD4 [47] 143, miR-375 and miR-339) in ESCC. There were also a number of
miR-373 ESCC LATS2 [83]
candidate miRNA studies in ESCC. Tian et al. [28] investigated that
Oncosuppressor microRNAs miR-10b was overexpressed in 95% of ESCC tissues. MiR-17-92
let-7 ESCC HMGA2 [57] cluster was found to be up-regulated in 75% of ESCC samples [29].
miR-34a ESCC NF-kB [69] MiR-31 was overexpressed in 78% of the ESCC tissues [30]. MiR-29c
miR-133a ESCC CD47 [84]
level was remarkably lower in ESCC tumor tissues and cell lines
miR-150 ESCC ERK-5, ZEB-1 [85]
miR-375 ESCC PDK-1, IGF1R [60,61]
compared to normal esophageal epithelia [31]. MiR-210 was
miR-205 ESCC ZEB-2 [86] down-regulated in ESCC and derived cell lines, particularly in
miR-145 ESCC FSCN-1 [66] poorly differentiated carcinomas [32]. In summary, the consis-
miR-29c ESCC CCNE [31] tently overexpressed miRNAs in ESCC, such as miR-21, miR-25,
miR-210 ESCC FGFRL1 [87]
miR-151, and miR-10b, and down-regulated miRNAs such as
Chemoresistance microRNAs miR205, miR-203, miR-145, miR-100, miR-99a, miR-27b, miR-
miR-141 ESCC YAP-1 [75] 125b, miR-133a, miR-133b, miR-143 and miR-375, were introduced
miR-148a ESCC, EAC CDC25B, BCL-2 [88,89] [22].
mir-296 ESCC Cyclin D1, p27, P-gp, Bcl-2, Bax [24,71]
miR-27a ESCC P-gp, Bcl-2, Bax [78,90]
miR-214 ESCC PTEN [91]
4. MiRNAs as biomarkers in esophageal cancer

EAC: Esophageal adenocarcinoma, ESCC: Esophageal squamous cell carcinoma,

ANXA1: Annexin A1, CDH1: Cadherin1, PDCD4: Programmed cell death 4, KLF4:
Kruppel-like factor 4, LATS2: Large tumor suppressor homolog 2, PDK-1:
Phosphoinositide-dependent kinase 1, IGF1R: Insulin-like growth factor-1 receptor,
FSCN-1: Fascin homolog 1, FGFRL1: Fibroblast growth factor receptor like- 1, YAP-1:
Yes-associated protein-1, PTEN: Phosphatase and tensin homolog.
the malignant development of BE and therefore may become the
useful biomarkers for malignant development of the BE patients.
Kan et al. [23] have also examined a miRNA signature in Barrett’s
tumorigenesis. In a study of 22 normal, 24 BE and 22 EAC tissue
samples, they determined 3 miRNAs that were up-regulated and 4
miRNAs that were down-regulated in BE or EAC compared to the
normal epithelium. The theory that the 3 up-regulated miRNAs
would have an oncogenic function is supported through the
finding that inhibitors of miR-25, miR-93 and 106b reduced the in
vivo carcinogenesis in mice. Interestingly, miR-25, miR-93 and
miR-106b were not remarkably up-regulated in BE relative to NE
tissues, but they were significantly up-regulated in EAC relative to
NE and BE. Again, this demonstrates that there is a progressive
participation of these miRNAs in the neoplastic process. In
summary, a number of miRNAs emerged as critical players in
the pathogenesis of EAC and may become the useful biomarker of
EAC and/or BE. MiR-21, miR-192, miR-194 were the most
consistently upregulated miRNAs in EAC, whereas miR-203,
miR-205, miR-100, miR-125b, miR-99a, miR-27b and let-7c were
consistently downregulated in EAC. Moreover, for miR-21,
miR-192, miR-203, miR-205, and let-7c, there appeared to be a
gradual increase or decrease of expression from NSE to BE to
EAC [22].
3.2. Dysregulation of miRNAs in ESCC development
ESCC is the prominent histological subtypes in Far East and all of
the published studies evaluating miRNAs in ESCC have come from
Asia [24,25]. Faber et al. [6] found that miR-21, miR-93, and miR-
342 were up-regulated whereas miR-203, miR-205, miR-27b, miR-
100, miR-125b, and let-7c were down-regulated in ESCC. Lee et al.
[26] discovered that four miRNAs, let-7d, miR-330, miR-340, and
miR-373, were overexpressed by at least 1.5-fold in each of the 5
In order to utilize miRNAs as the diagnostic tools for esophageal
cancer, varying levels of expression must be found in malignant
cells compared to normal or pre-malignant cells; therefore,
specific miRNAs that are remarkably up-regulated or down-
regulated between these groups must be determined [33]. MiR-
15b, miR-21, miR-203, miR-486-5p and let-7a, has been utilized for
the forecast of BE progression to EAC; it is likely that these miRNAs
are involved in the development of adenocarcinoma [34]. MiR-196
has also been discovered to increase progressively from normal
squamous mucosa to LGD and then HGD to adenocarcinoma [35].
In ESCC, miR-34b and miR-139 are found to be consistently
dysregulated, while miR-129 has been determined as a remark-
able diagnostic factor. MiR-34b can be down-regulated in ESCC
cells compared to normal ones [36]. Upregulation of miR-25, miR-
151 and miR-424 as well as down-regulation of miR-29c, miR-99a,
miR-100 and miR-140* are capable to forecast the squamous cell
carcinoma compared to adjacent normal tissues [37]. MiR-200b/c,
miR-205 and miR-429 in ESCC, are remarkably higher, and their
fluctuation has been validated. Therefore, they can be not only
biomarkers for ESCC, but could also be the tumor invasion
markers [38].
Recent researches have revealed that tumor derived miRNAs are
resistant to endogenous ribonuclease activity so it can be present
in human serum in a significantly stable form [39]. In addition, the
expression level of serum miRNAs is reproducible and consistent
among individuals [39,40]. These tumor-derived miRNAs are
present in the circulating blood at levels sufficient to be
measurable as biomarkers for the detection of tumors [39].
Zhang et al. [19] compared the pooled serum samples by Solexa
sequencing and determined 25 miRNAs atypically expressed
between the samples of ESCC patients and the normal ones. A
panel of 7 serum miRNAs (miR-10a, miR-22, miR-100, miR-127-3p,
miR-133a, miR-148b, and miR-223) was determined as a potential
biomarker for ESCC. The serum level of miR-200c can be useful for
predicting the responses to chemotherapy and the prognosis of
patients with EC who receive neo-adjuvant chemotherapy [41].
The up regulation of miR-21 and down regulation of miR-375 in
plasma samples of ESCC patients compared to normal subjects,
offered that the plasma level ratio of miR-21 to miR-375 could
 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519 513

reveal a better diagnostic biomarker than either markers alone.
Besides, the same Japanese group also assessed the prognostic
value of miR-21 and miR-375 and reported that the high plasma
level of miR-21 is associated with higher risk of reoccurrence and
poorer survival, whereas the high plasma level of miR-375 is the
symptom of better survival [42,43].
5. Oncogenic miRNAs in esophageal cancer
5.1. MiR-21
MiR-21 is the most routinely upregulated miRNA in human
cancers that shows the most constant association with both EAC
and ESCC progression as well as the prognosis of ESCC. MiR-21
regulates a plethora of target genes that are involved in cellular
survival, apoptosis and cell aggressiveness, including a number of
tumor-suppressor genes such as tropomyosin-1 (TPM1), PTEN,
maspin, and programmed cell death 4 (PDCD4) [44,45] (Fig. 2a).
Down-regulation of miR-21 can suppress the cell proliferation and
induce apoptosis, with identified direct targets such as FASL, TIMP3
and RECK [46]. Moreover, the over-expression of miR-31 and
miR-21 may be associated with the down-regulation of their
respective tumor suppressor targets PPP2R2A and PDCD4 in ESCC.
MiR-31 and miR-21 levels are directly related to the appearance of
ESCC [47].
dependent G1/S cell cycle arrest in response to DNA damage [22].
Furthermore, miR-10b acts as oncomiR and its oncogenic function
is at least relatively mediated by KLF4 in ESCCs [50].
5.3. MiR-17-92 polycistron
MiR-17-92 cluster, also called as oncomir-1, is one of the
oncogenic miRNAs that is found overexpressed in malignant
cancers. The gene is settled at 13q31.3, encodes six mature miRNAs
including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and
miR-92-1, and has been involved in the oncogenesis of multiple
malignant cancers [29]. The miR-17-92 cluster was discovered to
be overexpressed in 21 out of 28 (75%) esophageal cancer samples.
In addition, overexpression of the miR-17-92 cluster could elevate
the cellular growth in vitro and in vivo, and the suppression of miR-
19a, induced apoptosis in vitro and impaired tumor growth in vivo.
TNF-a (tumor necrosis factor-a) was verified as a direct target of
miR-19a and TNF-a-encoded mRNA includes a 30 UTR element that
is relatively complementary to miR-19a [29]. In addition, miR-92a
was found to regulate the migration and invasion but not apoptosis
of esophagus squamous carcinoma cell lines in vitro. Furthermore,
miR-92a directly targeted CDH1 and suppressed the expression of
CDH1 [51].
5.4. MiR-200c and miR-221/222

5.2. MiR-10b Dysregulation of miR-200c was discovered in several types of

MiR-10b is overexpressed in various types of cancer [48,49]
including ESCC. Tian et al. [28] found that the cellular miR-10b
expression level is correlated with the cell motility and aggres-
siveness in various human ESCC cell lines. Furthermore, they
identified KLF4 (Krüppel-like factor 4), a known tumor suppressor
gene that has been shown to suppress esophageal cancer cell
migration and aggression, as a direct target of miR-10b. KLF4
regulates the expression level of p21 and mediates the p53-
cancer, including esophageal cancer [52]. Studies have revealed a
correlation between miR-200c expression and resistance to
anticancer drugs. PI3 K/Akt signaling pathways are disrupted in
majority types of tumor, such as ESCC [53] that affect their
responsiveness to chemotherapy [54]. Consequently, miR-200c
influences the responsiveness to chemotherapy and radiotherapy
in ESCC, and patients with high expression of serum miR-200c may
endure higher risk of death; thereby the serum level of miR-200c
can be useful for anticipating responses to chemotherapy [52]. In

Fig. 2. MicroRNAs in cell proliferation and cell death. MicroRNAs controlled the cell destiny by regulating cell proliferation and cell death pathways with their oncogenic and
tumor suppressive function. (a): Role of OncomiRs in cell death and cell proliferation. (b): Role of Tumor suppressors in cell death and cell proliferation. KLF-4: Krüppel-like
factor-4, CDX2: Caudal-related homeobox 2, IGF1R: Insulin-like growth factor-1 receptor, FSCN1: Fascin-1.
514 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519
addition, miRNA-221/222 acts as an oncogene in EAC. The raised
levels of miR-221/222 have also been observed in EAC. The
exposure of esophageal cells to bile acids may activate farnesoid X
receptor and upregulate the levels of miR-221/222, by decreasing
the levels of p27Kip1 and promoting the degradation of CDX2.
Consequently, down-regulation of p27Kip1, as a result of the
increased expression of miR-221/222, increased the degradation of
Cdx2 and cell proliferation during the exposure to bile acid in
human esophageal epithelial cells. This mechanism might play a
crucial role in the development of esophageal adenocarcinoma
[55].
6. Tumor suppressor miRNAs in esophageal cancer
6.1. Let-7
Let-7 was remarkably, discovered as the second miRNA after
lin-4 in C. elegans and known as one of the most extensive miRNA
[56]. High mobility group A2 (HMGA2) protein, is a non-histone
DNA binding factor that is highly expressed during embryogenesis
and overexpressed in different benign and malignant cancers
[57,58]. There are several let-7 complementary regions at the
30 -UTR of HMGA2 and the role of let-7 is significant in regulating
HMGA2 protein expression to prevent the cell proliferation in ESCC
(Fig. 2b). In addition, HMGA2 is negatively regulated by let-7 at the
post-transcriptional level [57]. Also, the expression of let-7 in
esophageal cancer could be utilized to forecast the sensitivity/
resistance to cisplatin-based chemotherapy, and studies showed
that the high expression of let-7c is remarkably correlated with the
clinical response in esophageal cancer. Let-7c expression modu-
lated apoptosis in cisplatin-treated cells, through the down-
regulation of IL-6– mediated signaling pathway. Moreover,
phosphorylated STAT3, which is the downstream of IL-6, was
induced by cisplatin in esophageal cancer cells. Transfection of let-
7c can directly suppress the cisplatin-activated interleukin IL-6/
STAT3 pro-survival pathway after the genotoxic chemotherapy in
esophageal cancer cells [59].
6.2. MiR-375
The promoter of miRNA-375 is regularly hyper methylated in
esophageal cancer, and miR-375 negatively regulates the 3-
phosphoinositide dependent protein kinase-1 (PDK1) in EC [60].
In addition, the frequent promoter hyper-methylation of miR-375
was discovered in ESCC tissues [61]. Moreover, miR-375 could
interact with the 30 UTR of IGF1R and down-regulate its expression
in vitro. Additionally, miR-375 expression level was negatively
correlated the expression of IGF1R in clinical samples [61]. MiRNA-
375 can be restored through the cyclic hydroxamic acid-containing
peptide 31, a histone deacetylase inhibitor of ESCC cells. LDHB and
AEG-1/MTDH are detected as miR-375-targeted genes, with both
mRNA and protein expression levels of these verified in ESCC
clinical specimens [62]. These data prepare compelling evidence
supporting that the down-regulation of miR-375, by promoter
hyper-methylation, is one of the molecular mechanisms implicat-
ed in the development of ESCC [22].
6.3. MiR-145
MiRNA-145 is down-regulated in various cancers [63] and
precancerous lesions [64]. Particularly, Sachdeva et al. [65]
demonstrated that c-Myc is a direct target for miR-145. Transcrip-
tionally, miR-145 is induced by the tumor suppressor p53.
Moreover, miR-145 provides a direct connection between p53
and c-Myc. The loss of heterogeneity (LOH) of 17p13 (p53 locus)
and the amplification of 8q24 (Myc locus) are both frequent
molecular modifications in ESCC and EAC, offering that miR-145
may show its tumor suppressor function in EC through the
p53-Myc network. However, there has not been any direct
evidence exhibiting that miR-145 prevents c-Myc in EC. On the
other hand, Kano et al. [66] determined a novel target gene, FSCN1
(actin-binding protein, Fascin homolog 1), for miR-145. FSCN1 was
overexpressed in ESCC tumors compared to normal epithelium,
and FSCN1 overexpression was remarkably connected to the extent
of the tumor, lymph node metastasis, and poor prognosis [67].
These data offer inhibitions of FSCN1, which may be one of the
mechanisms for the tumor suppressor function of miR-145 in ESCC.
6.4. MiR-34a
MiR-34a has recently been found to act as a critical tumor
suppressor in the progression of different cancers. Some of the
genes referring to the cell cycle and apoptosis control including
CDK4/6, cyclin D1, E2F3, MYCN, SIRT1 and Bcl2 are shown to be
downregulated by miR-34a [68,69]. Although the most critical
regulator of miR-34a expression is the well-known tumor
suppressor p53 [70], bioinformatics analysis demonstrated that
there were some potential NF-kB binding sites located in the
promoter region of miR-34a gene. A novel mechanism of miR-34a
regulation in ESCC is that NF-kB can elevate the miR-34a
expression levels by the direct binding to its promoter, and the
wild-type p53 is responsible for NF-kB- mediated miR-34a
transcriptional function. This might show a novel perspective
for the roles of miR-34a and NF-kB in esophageal cancer
development [69].
7. Chemoresistance miRNAs in esophageal cancer
7.1. MiR-148a
MiRNA-148a is one of the drug resistance related miRNAs in EC.
The expression levels of miR-148a in EAC are inversely related to
the cancer differentiation [71]. In patients with locally advanced
ESCC, the expression of miR-148a is associated with the disease
recurrence and the tumor-related mortality [37]. Hummel et al.
discovered that miR-148a could improve the response to
chemotherapy in the responsive and resistant EC cells [72].
Upregulation of miR-148a could remarkably increase the sensitiv-
ity of EC cell lines to chemotherapy, represented by a reduction in
the cell viability after 5-fluorouracil (5-FU) treatment. Upregula-
tion of miR-148a can sensitize the chemotherapy-sensitive
esophageal cancer cell lines to cisplatin (CDDP) and decrease
the resistance in chemotherapy resistant variants (Fig. 3a). There is
a tendency toward a better response to 5-FU in the 5-FU-
responsive and resistant cells following the miR-148a transfection.
The exact implicated mechanisms should be examined by further
experimental and clinical studies [71].
7.2. MiR-141
MiR-141 is located on chromosome 12 and has a particular
pattern of expression in human cancers of epithelial cell type. The
expression of miR-141 is attenuated in Barrett’s epithelium when
compared with gastric and duodenal epithelia [73]. MiR-141 may
also be implicated in the progression of gastric cancer through its
inhibitory effect on cell proliferation [74]. Imanaka et al. discovered
that miR-141 has a critical regulatory function in the development
of CDDP-resistance in CDDP-resistant ESCC [75]. MiR-141 is highly
expressed in the CDDP-resistant EC cell lines. When expressed
ectopically in the CDDP-sensitive cell lines, the cell viability after
CDDP treatment is remarkably augmented. MiR-141 may directly
target the 30 untranslated region (UTR) of YAP1, which plays an
 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519 515

Fig. 3. Effect of microRNAs in sensitivity/resistance to chemotherapy. a) Up-regulation of miR-148a remarkably decreases resistance of EC cell lines to chemotherapy by
targeting CDC25B and BCL-2. Also, up-regulation of miR-141 increases drug resistance through targeting the 30 untranslated region (UTR) of YAP1. YAP-1: Yes-associated
protein 1, BCL-2: B-cell lymphoma 2, P-gp: P-glycoprotein. b) Down-regulation of miR-27a and miR-296 may promote ADR-induced apoptosis through regulation of Bcl-2,
Bax, and p-gp.

important role in the DNA damaging agents-induced apoptosis,
and thus down-regulates the YAP1 expression (Fig. 3a). Moreover,
miR-141 may play a crucial regulatory role in the progression of
CDDP resistance in ESCC [71,75].
7.3. MiR-27a
augmented accumulation of ADR. Down-regulation of miR-27a can
remarkably attenuate the expression of P-gp, Bcl-2, and the
transcription of the MDR gene 1, as well as up-regulate the
expression of Bax, and confer the drug-induced apoptosis through
enhancing the Bcl-2/Bax ratio, thereby effectively reverse the drug
resistance of esophageal cancer cells [78] (Fig. 3b).

MiR-27a is highly expressed in cancer cells and may act as an
oncogene through the regulation of cell survival and angiogenesis
[76]. MiR-27a may repress the cdc2/cyclin B inhibitor and thereby
facilitate the cancer cell proliferation by arresting cells at G2-M
[77]. MiR-27a is capable of regulating the cell cycle progression and
angiogenesis following the drug treatment, and delicately cooper-
ate with several other gene regulators to ensure the establishment
of drug defense network [78].
Down-regulation of miR-27a may confer the sensitivity of both
P-gp-related and P-gp-non-related drugs on EC cells, and may
elevate Adriamycin (ADR)-induced apoptosis, accompanied by
7.4. MiR-296
MiR-296 is implicated in many physiological and pathological
processes, such as carcinogenesis, fetal alcohol syndrome, insulin
production as well as insulin secretion [79,80]. Inhibition of
miR-296 with antagomirs may attenuate angiogenesis in tumor
xenografts in vivo. Additionally, miR-296 reveals low levels of
expression in the NIH3T3 cells after the UVB irradiation [81],
determining that miR-296 may be implicated in DNA damage
repair. Reduced expression of miR-296 is capable to differentiate
long term survivors with the node-positive esophageal cancer
516 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519
from those dying within 20 months. Down-regulation of miR-296
may prevent the growth of EC cells in vitro and in vivo through
regulation of cyclin D1 and p27. Down-regulation of miR-296 can
confer sensitivity of drugs on EC cells by attenuating the releasing
amount of Adriamycin (ADR) through the regulation of P-gp [24].
Additionally, down-regulation of miR-296 may promote ADR-
induced apoptosis through the regulation of Bcl-2 and Bax.
Consequently, miR-296 may play crucial roles in the pathogenesis
of EC and is considered as a potential target for the intervention of
this malignancy [71] (Fig. 3b).
8. Potential in screening and treatment of EC
Until now, endoscopic biopsy and histopathological examina-
tions are the golden standards for high-risk subject screening and
early detection for EC worldwide. We can only determine 1–2
percent early carcinomas and 15–20 percent precancerous lesions
in asymptomatic population aged higher than 35 years in high-
incidence areas of EC through endoscopic biopsy and histopatho-
logical examinations [92]. Yet, nearly 80% asymptomatic popula-
tions were in the normal range. Thus, there exists over endoscopic
application.
Additionally, standard sterilizing procedure also restricts the
wide application of endoscopy in asymptomatic population. In
addition, the projected cost of mass screening in China for
detection of cancers is increasing annually. An urgent and critical
problem to be solved is to attenuate the blindness in the screening,
minify the range of endoscopy and promote the detection
sensitivity of early carcinoma in high-risk subjects [3]. Easily
accessible and minimal invasive biomarkers for EC and for other
diseases are much valuable. The PCR technique for detection and
quantification of miRNA [93] in the blood would be one of the
promising tools of screening individuals for EC and other cancers
because of its universal application, suitable management, low cost
and high sensitivity. Therefore, miRNAs acting as oncogenic or
performing tumor suppressor activities, are novel biomarkers for
EC therapies [3].
9. MiRNAs as therapeutic targets in esophageal cancer
There have been many innovative studies on miRNAs and EC
treatment. MiRNAs are exceptional candidates for novel molecular
targeting therapies according to their capability to regulate
multiple genes in molecular pathways. The reason for utilizing
miRNAs as potential anti-cancer drugs is based on two major
findings: first, miRNA expression can be dysregulated in cancer;
second, targeting miRNA expression may alter the cancer
phenotype [33]. In esophageal cancer patients, the response to
chemotherapy (both neo-adjuvant and adjuvant) for similar cancer
phenotypes, varies widely from one patient to another [93], and
Table 2
Therapeutic function of miRNAs in esophageal cancer.
Therapeutic Function
Improves the sensitivity of esophageal cancer cell to cisplatin or 5-fluorouracil.
Confers the resistance to cisplatin-induced apoptosis by targeting YAP-1.
Down-regulation of miR-27a, enhance the response to anti-tumor drugs and promote cell apoptosis
Down-regulation of miR-296, enhance the response to anti-tumor drugs and promote cell apoptosis.
Predictive markers for multimodality therapy of patients with locally advanced EC.
Predictive markers for multimodality therapy of patients with locally advanced EC.
Organometallic arene Ru (II) drug Rawq01 prevent the PI3K-AKT pathway through the miR-21 inhibition.
Blocked with AMOs.
Blocked with AMOs.
Blocked with AMOs.
Blocked with AMOs.
AMOs: Anti-miRNA oligonucleotides.
there is an evidence of aberrant miRNA expression being
implicated in regulating the expression of multi-drug resistance
(MDR)-related genes [94]. Furthermore, several miRNAs have
recently been associated with various esophageal tumor responses
to therapy (Table 2). For instance, miR-148a expression remarkably
improves the sensitivity of esophageal cancer cell lines to cisplatin
or 5-fluorouracil [72], and miR-141 confers the resistance to
cisplatin-induced apoptosis by targeting YAP1 [75]. In addition, the
decreased miR-27a and miR-296 expression levels enhance the
response to anti-tumor drugs and promote cell apoptosis [24,78].
According to results of microarray analysis, miR-192, miR-194
and miR-622 have been selected, and expressions of these three
miRNAs have been revealed to be remarkably diminished after the
neo-adjuvant therapy, confirming the array profiling data.
Significantly, pre-therapeutic intratumoral expression of both
miR-192 and miR-194 is remarkably related to histopathological
response of ESCC to the multimodal therapeutic treatment.
Consequently, miR-192 and miR-194 can be considered to be
predictive markers for the therapeutic response in multimodality
therapy of patients with locally advanced EC [95]. As one of the
most commonly dysregulated miRNAs in many cancers, miR-21 is
remarkably related to the pathological stage of ESCC. The impact of
miR-21 on PTEN/AKT signaling pathway has been found to be
abrogated by the novel organometallic arene Ru (II) drug Rawq01.
Rawq01 can upregulate the expression of PTEN through the miR-21
inhibition and therefore prevent the PI3K-AKT pathway [95].
The in vivo stability of miRNAs has been successfully utilized in
preclinical models of miRNA-based treatments (either alone or
combined with conventional and/or targeted therapies) [94]. Two
main strategies are prevalently focusing on miRNA expression
[94,96]: 1) applying oligonucleotides (or virus-based constructs) to
block oncogenic miRNA expression or replace the loss of tumor
suppressor miRNAs, 2) regulating miRNA expression through
targeting their transcription/processing. Unluckily, both the issues
of tissue-specific delivery and the cellular uptake of synthetic
oligonucleotides are still major obstacles to the progression of
miRNA targeted therapies. Therefore, various chemical modifica-
tions in oligonucleotides have been explored (i.e. morpholinos,
peptide nucleic acids, cholesterol conjugation, and phosphoro-
thioate backbone modifications) [94,97]. In summary, there are
two feasible approaches for utilizing miRNAs as the cancer
therapeutic factors, either through antisense-mediated inhibition
of oncogenic miRNAs or by ‘replacement’ of under-expressed
tumor suppressive miRNAs with miRNA mimetic or viral vector-
encoded miRNAs [93].
Evident inhibitory types of molecule to miRNA are anti-miRNA
oligonucleotides (AMOs), which may inhibit interactions between
miRNA and the target mRNAs through competition [98]. Utilizing
AMOs is intended to block the expression of an oncogenic miRNA,
including miR-17-29 cluster, miR-21, miR-122, miR-155 and miR-
MiRNAs
miR-148a
miR-141
miR-27a
miR-296
miR-192
miR-194
miR-21
miR-17–29 cluster
miR-122
miR-155
miR-221
 M. Hemmatzadeh et al. / Biomedicine & Pharmacotherapy 82 (2016) 509–519
221. In addition, the miR-mask, a sequence with excellent
complementarity to the binding site for an endogenous miRNA
in a target gene, can also prevent the access of endogenous miRNA
to its binding site [99]. Moreover, restoring the endogenous
complement of miRNAs in a cell can be attained by either
introducing a short synthetic duplex RNA molecule that is loaded
into the RISC or by motivating the expression of the stem-loop pre-
miRNA in a viral vector expression system or the miRNA mimetic
system, which mimics the endogenous mature miRNA, including
let-7, miR-15 and miR-34 [100].
10. Conclusion
Many comprehensive miRNA expression profiling studies have
been appeared in EAC and ESCC tissues. MiRNAs clearly play
critical roles in the pathogenesis of EAC and ESCC. A number of
consistently dysregulated miRNAs have been determined in EC,
such as upregulation of miR-21, miR-31, miR-221/222, miR-17-92
polycistron, miR-10b, miR-200c, and miR-373, and down-regula-
tion of miR-375, miR-203, let-7, miR-145, miR-34a, miR-143, miR-
210, and 29c. Consequently, the altered miRNA expression
patterns may exert a remarkable effect on the balance between
the tumor suppressors and oncogenes. The field of miRNA
research is very promising in the area of cancer research and
therapy, and the pharmaceutical companies have been trying to
accelerate the progress of therapeutic miRNA technologies. The
finding that miRNAs play a critical role in MDR of EC has laid the
foundation for potential effective clinical management of EC
cases. The finding of drug resistance-related miRNAs in EC has
helped to show novel mechanisms in MDR. Continuous expansion
of determined miRNAs as biomarkers and therapeutic targets may
suggest the fascinating insights into the complexity of gene
expression regulation. In addition, performing research into
miRNA participation in esophageal carcinogenesis is a rewarding
field, and studying altered miRNA expression patterns can suggest
substantial insights into the pathogenesis of this devastating
disease. Further work to determine the down-stream targets of
these miRNAs will improve our understanding of their mecha-
nisms of action, which will shed new light on utilizing them
clinically as biomarkers or therapeutic targets in esophageal
cancer.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
None.
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