Evaluation of miR-21 and miR-375 as prognostic biomarkers in oesophageal cancer in high-risk areas in China
Clin Exp Metastasis
Evaluation of miR-21 and miR-375 as prognostic biomarkers in oesophageal cancer in high-risk areas in China
Yutong He 0 1 2
Jing Jin 0 1 2
LiQun Wang 0 1 2
Yuejiao Hu 0 1 2
Di Liang 0 1 2
Huichai Yang 0 1 2
Yueping Liu 0 1 2
Baoen Shan 0 1 2
0 Pathology Department, The Fourth Hospital of Hebei Medical University/The Tumor Hospital of Hebei Province , Shijiazhuang , China
1 Hospital Medical Insurance Department, The Fourth Hospital of Hebei Medical University/The Tumor Hospital of Hebei Province , Shijiazhuang , China
2 Cancer Institute, The Fourth Hospital of Hebei Medical University/The Tumor Hospital of Hebei Province , Shijiazhuang 050011 , China
MicroRNAs have been associated with prognosis in oesophageal cancer (EC), suggesting that miRNAs could play a role in guiding treatment decisions. The aim of this study was to evaluate the prognostic potential of miRNAs found to be associated with zinc deficiency in a geographical area with a high incidence of EC. miRNAs found to be associated with zinc deficiency were isolated from EC cell lines cultured with various Zn levels. The expression levels of the miRNAs were quantified using qRT-PCR. The potential prognostic value of the selected miRNAs was assessed in a cohort study of 88 patients from an area in China with a high incidence of EC. Correlations between miRNAs and patient characteristics were assessed using v2 statistical tests or Fisher's exact test. A Cox proportional hazards model was used to assess the correlations between miRNAs and overall survival (OS). Forest plots were performed to evaluate the prognostic impact of the miRNAs examined in the present study in the Asian population. The expression levels of miR-21, miR-31, miR-93 and miR-375 were different when Zn levels were varied in EC cell lines, but only miR-21 and miR-375 were associated with patient characteristics and prognosis in patients with EC from an area of China with a high incidence of EC. The patients expressing high levels of miR-21 had poor OS (HR 2.15, 95% CI 1.16-3.97), whereas those with high levels of miR-375 had improved OS (HR 0.47, 95% CI 0.26-0.87).The patients with both a high level of miR-375 and a low level of miR-21 had significantly better outcomes. Forest plots based on an analysis of this Asian population indicated that a high level of miR-21 significantly predicted a shortened OS (HR 1.83, 95% CI 1.42-2.37), whereas a high level of miR-375 was significantly correlated with increased survival (HR 0.56, 95% CI 0.43-0.73). MiR-21 and miR-375 could be used as prognostic biomarkers in areas with a high incidence of EC, and combining these markers may results in a better effect.
Oesophageal cancer; miR-21; Prognostic
-
& Baoen Shan
Oesophageal cancer (EC) is one of the most common
cancers in the world. With an estimated 456,000 new cases
and 400,000 deaths per year, EC had the 8th highest
incidence of all cancers and the 6th highest cancer-related
mortality rate worldwide, based on GLOBOCAN 2012.
Approximately 223,000 new cases and 197,200 deaths are
associated with EC in China alone.EC therefore accounts
for 52.8% of all cases and 49.3% of deaths from
oesophageal malignancies worldwide [1]. The incidence of EC
varies widely between different regions. For example, it
ranges from 3 per 100,000 in low-incidence regions to over
100 per 100,000 in high-incidence areas. Cixian, in Hebei
Province, is an area with one of the highest rates of EC in
China and the world [2]. Although postoperative recovery
and survival rates have substantially improved as a result of
continuous progress in diagnostic technologies and
therapies, the survival rate remains unsatisfactory, with an
approximately 20% overall 5-year survival rate [3]. A
multitude of factors influence a prognosis of EC. These
include the pathological stage, histological type,
pathogenic sites, and biological behaviours of the cancer and the
measures used to treat it. However, the influence of some
factors has not yet been explored. Because there have been
discrepancies in the results of studies examining cancers
with the same stage, there is an urgent need for new
parameters that can complement differentiation and TNM
staging to enable more accurate predictions of prognoses
and to provide the best preoperative counselling to patients.
To obtain more information regarding the characteristics
and survival prospects of patients with ESCC, we focused
on the influence of a particular class of microRNAs
(miRNAs) on the abnormal expression of genes or proteins
[4].
miRNAs are a species of small, conserved endogenous,
noncoding, single-stranded RNAs that are approximately
22 nucleotides in length. They act as post-transcriptional
regulators by targeting mRNA for degradation or
translational repression, usually resulting in gene silencing [5].
They are known to possess the ability to regulate important
cellular processes, such as differentiation, the cell cycle,
proliferation and apoptosis. By affecting these processes,
they can act as tumour suppressors or oncogenes in a
variety of cancers. Recent studies have indicated that
almost 50% of miRNAs target tumour-associated sensitive
sites in genomes, where they can function as either
oncogenes or tumour-suppressor genes by recognizing and
combining with targeted molecules and either inducing the
degradation of the mRNA or post-transcriptionally
regulating the translation of the mRNA [6]. A growing amount
of evidence suggests that the expression levels of miRNAs
in cancer tissues are useful prognostic markers, and
changes in the miRNA expression profiles of cancers are being
intensively studied in EC. These include miR-16 and
miR21, which are predictors of poor survival in EC [7].
The most common features of regions with a high
incidence of EC are an under-developed economy and low
nutrient intake. Zinc is an essential trace element, and zinc
deficiency (ZD) is a common phenomenon in areas with a
high incidence of EC. An inflamed ZD oesophagus has a
distinct miRNA signature that resembles the human EC
miRNA signature [8]. Louise et al. reported that in
individuals with marked ZD, the oesophagus had a
15-microRNA signature in which miR-21, miR-31, and miR-223
were the most up-regulated species [9]. In this study, we
examined the levels of a number of miRNAs that are
associated with altered Zn levels in EC cell lines and
analysed the associations between miRNA levels and
survival in 88 patients from an area with a high incidence
of EC to explore the possible utility of using ZD-related
miRNAs as prognostic biomarkers for EC in high-risk
areas.
Materials and methods
Human esophageal cancer cell line KYSE170 was obtained
from MD Anderson Cancer Center in the United States.
The cell line Eca109 was obtained from China Infrastruture
of Cell Line Resources. KYSE170 and Eca109 were
divided into two groups. One group was cultured in RPMI
1640 medium (Gibco, USA) with 10% foetalbovine serum
(FBS, Gibco, USA), and the other was cultured in RPMI
1640 medium with 10% FBS plus ZnSO4. We tried 4 levels
of ZnSO4 (25, 50, 75 and 100 uM/L) for preliminary
experiment and chose 50 uM/L in this experiment
according to two principles: (1) the level of ZnSO4 had no
effect on cell status; (2) the level of ZnSO4 made most
significant changes in miRNA levels. The expression levels
of miRNAs were determined for each cell line to analyse
the effect that Zn levels had on the miRNAs. The miRNAs
that were measured included miR-21, miR-31, miR-93,
miR-193a, miR-193b, miR-342 and miR-375, and their
relationships with Zn levels were determined using Target
Scan Human.
Patients and samples
This analysis was performed using data obtained from 92
consecutive patients from Cixian, which is a region in
Hebei Province with a high rate of histologically confirmed
EC. All patients were surgically treated at The Fourth
Hospital of Hebei Medical University (also known as The
Tumour Hospital of Hebei Province, which is a large and
comprehensive level three, grade A hospital) from January
1, 2007 to December 31, 2008. The inclusion criterion was
that the patient must have received a pathological diagnosis
of primary EC. Surgical samples were obtained within 30
minutes post-operative from each patient and immediately
stored in liquid nitrogen. A total of 92 cases that were
admitted met the inclusion criterion and only 88 cases with
completed follow-up evaluations were included in the
study. The follow-up rate was 95.65%. Among these 88
cases, 46 died of EC, whereas 42 were alive on March 15,
2014. The follow-up period ranged from 1 to 88 months,
and the median follow-up period was 62 months. Overall,
69 cases were male (78.4%), and 19 cases were female
(21.6%). The median age was 67 years old (range
50–87 years old). In addition, the serum zinc levels of
patients and healthy controls were measured. This included
10 patients from high and 10 from low incidence areas of
EC. The patients were new cases in 2016. The controls
were healthy volunteers, including 10 from high and 10
from low incidence areas of EC. This study was approved
by the Institutional Human Ethics Committee, and prior
informed consent was obtained from all patients. The
baseline patient and tumour characteristics are summarized
in Table 1.
Data collection
Personal patient data were extracted from medical records
and included gender, age, occupation and family history.
Tumour data consisting of the pathogenic site, pathological
type, TNM stage and surgical situation were also obtained.
Follow-up studies
Follow-up evaluations were performed according to the
standard follow-up system of the hospital every 6 months
after patients were discharged from the hospital. The
deadline for follow-up evaluations was March 15, 2014.
All patients were followed from the date of histological
diagnosis until death or last day of follow-up and were
followed for at least 20 months or until death. The survival
Table 1 Patient and tumor characteristics
period was measured from the date of admission to the date
of death or to the date of the follow-up deadline.
Extraction and quantification of miRNA
Total RNA was extracted from tissue samples according to
the instructions in the manual form iRNAVanaTM
PARISTM (Ambion, TX, USA). miRNA levels were analysed
using qRT-PCR with TaqMan, miRNA reverse
transcription assays, and the appropriate primers according to the
manufacturer’s instructions. Briefly, 10 ng of total RNA
was used as the template for 15 lL reverse transcription
reactions. Probes were specially designed for specific
mature miRNAs. For each miRNA, reactions were
performed in triplicate using the 7500 RT-PCR system
(Applied Biosystems), and RNU66 (Applied Biosystems, cat.
no. 4373382) was used as the normalization control. The
sequence used as the forward primer for miR-16(a
housekeeping gene) in this study was 50-CAGCCTAGCAG
CACGTAAAT-30, the sequence used as the reverse primer
for miR-16 was 50-GAGGTATTCGCACCAGAGGA-30,
and the sequence used for the RT reaction was 50-GTC
TCCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGA
GGAGACCGCCAA-30.
The sequence of target gene:
miR-21-3p RT: 50-CTCAACTGGTGTCGTGGAGT
CGGCAATTCAGTTGAGACAGCCCA-30
miR-21-3p F: 50-ACACTCCAGCTGGGCAACACCA
GTCGATG-30
miR-31-3p RT: 50-CTCAACTGGTGTCGTGGAGTC
GGCAATTCAGTTGAGATGGCAAT-30
miR-31-3p F: 50-ACACTCCAGCTGGGTGCTATGC
CAACAT-30
miR-93-5p RT: 50-CTCAACTGGTGTCGTGGAGTCG
GCAATTCAGTTGAGCTACCTGC-30
miR-93-5p F: 50-ACACTCCAGCTGGGCAAAGTG
CTGTTCGT-30
miR-193a RT: 50-GTCGTATCCAGTGCGTGTCGTG
GAGTCGGCAATTGCACTGGATACGACACTGGG-30
miR-193a F: 50-GGCCAACTGGCCTACAAAGT-30
miR-193b RT: 50-GTCGTATCCAGTGCGTGTCGTG
GAGTCGGCAATTGCACTGGATCCGACTCATCT-30
miR-193b F: 50-GGCGGGGTTTTGAGGGCG-30
miR-342 RT: 50-GTCGTATCCAGTGCGTGTCGTG
GAGTCGGCAATTGCACTGGATACGACTCAATC-30
miR-342 F: 50-CCGGAGGGGTGCTATCTGT-30
miR-375-3p RT: 50-CTCAACTGGTGTCGTGGAGTC
GGCAATTCAGTTGAGTCACGCGA-30
miR-375-3p F: 50-ACACTCCAGCTGGGTTTGTTCGT
TCGGC-30
All R: TGGTGTCGTGGAGTCG
We searched PubMed, EMBASE, and the Central
Registry of Controlled Trials of the Cochrane Library up to
April 21, 2016. The following search strategy was
employed to both PubMed and EMBASE: microRNA/
miR-21, microRNA/miR-375, esophag*
cancer/carcinoma/neoplasm. No restrictions on publication type,
publication language, or publication year were applied.
Cited references from the selected articles were manually
searched and assessed. Original research studies that met
the following criteria were included: (1) miR-21,
miR375 was determined from human esophageal cancer tissue
by quantitative methods; (2) to explore the relationship of
miR-21, miR-375 and clinical prognosis of esophageal
cancer; (3) There are HR and 95% confidence intervals in
the literature or can be extrapolated from the literature. If
multiple studies had a duplicate patient cohort, only one
study was included for analysis. And the exclusion
criteria were: (1) the miR-21, miR-375 and clinical
prognosis of esophageal cancer were not studied in human
beings; (2) the HR and 95% confidence intervals couldn’t
be evaluated from the study; (3) the available outcomes
appear in a review.
Statistical analysis
The SPSS 21.0 and Excel software packages were used for
the statistical analysis. We used c2 tests or the Fisher’s
exact probability method to determine the relationships
between the miRNAs and patient information variables
(i.e., gender, age, family history, pathological type, TNM
stage, and surgery incidence).OS rates were calculated
using the Kaplan–Meier method. The OS rates of the
patients in different groups were compared using the
logrank c2 test (inspection level 5, 0.05). Univariate and
multivariate Cox regression models were used to determine
the factors that influenced OS.
Effects of Zn on miRNAs in EC cell lines
We compared the expression levels of miRNAs in human
EC cell lines that were cultured with or without Zn. We
used the KYSE170 and Eca109 cell lines to detect the
effects of Zn on the selected miRNAs. The expression
levels of the miRNAs in the cells incubated without Zn
were used as the standards, and the relative levels of
miR21, miR-31, miR-93 and miR-375 were significantly
different (P \ 0.05) in the cells incubated with Zn. The
expression levels of other miRNAs were not significantly
Fig. 1 a Expression levels of miRNAs in KYSE170 cells grown with
and without Zn. The cell line without Zn was cultured in RPMI 1640
medium with 10% foetalbovine serum, and the cell line with Zn was
cultured in RPMI 1640 medium with 10% FBS plus 50 lmol/L
ZnSO4. b Expression levels of miRNAs in Eca109 cells grown with
and without Zn. The cell line without Zn was cultured in RPMI 1640
medium with 10% foetalbovine serum, and the cell line with Zn was
cultured in RPMI 1640 medium with 10% FBS plus 50 lmol/L
ZnSO4
different (Fig. 1). And there was no differences in status
between the cells were cultured with or without Zn.
The serum zinc levels of patients and healthy volunteers
from high and low incidence areas of EC were measured.
The serum zinc level of EC patients from high incidence
areas (cancer high group, CH) was 81.00 ± 4.03 ug/
100 mL, from low incidence areas (cancer low group, CL)
was 83.90 ± 5.51 ug/100 mL; the serum zinc level of
control from high incidence areas (normal high group, NH)
was 87.20 ± 5.67 ug/100 mL, from low incidence areas
(normal low group, NL) was 111.10 ± 5.11 ug/100 mL.
We did a LSD analysis and found that the serum zinc level
of CH was lower than NH (P = 0.010), of CL was lower
than NL (P \ 0.001), of NH was lower than NL
(P \ 0.001) and of CH was no significant differences with
CL (P = 0.213).
Association of the miRNAs with patient
characteristics
We sought to determine the relationships between the
miRNAs for which expression levels were associated with
Zn levels, including miR-21, miR-31, miR-93 and
miR375, with patient characteristics and prognostic values. The
expression levels of the miRNAs were determined using a
microarray analysis that separated the miRNAs into groups
of high and low expressors based on the mean relative
cutoff value for each miRNA. The results showed that only
miR-21 and miR-375 were associated with patient
characteristics and prognoses.
Correlations between miR-21 and patient characteristics
were assessed using the v2 statistical test or Fisher’s exact
test. The results showed that miR-21 expression was
associated with pathogenic sites (P = 0.037): high levels
of miR-21 were correlated with upper sites but not with
gender (P = 0.229), age (P = 0.722), pathological type
(P = 0.191), TNM stage (P = 0.618) and Postoperative
metastasis (P = 0.349) (Table 2). The same test was used
to analyse the correlations between miR-375 levels and
gender (P = 0.506), age (P = 0.704), pathological type
(P = 0.708), TNM stage (P = 0.845), Postoperative
metastasis (P = 0.190), none of which were correlated
with miR-375 expression. However, the pathogenic site
was associated with miR-275 in that low miR-375
expression was correlated with upper sites (P = 0.022)
(Table 2).
Prognostic value of miR-21 and miR-375 in EC
A univariate Cox proportional hazards model indicated that
both miR-21 (P = 0.032) and miR-375 (P = 0.028) were
significantly correlated with OS. The pathogenic site
(P = 0.034) and TNM stage (P = 0.013) were also found
to be significantly associated with OS (Table 3). The
univariate analysis showed that when evaluating OS, better
outcomes were observed in EC patients with low miR-21
expression and that patients with high miR-21 expression
had worse outcomes (Fig. 2a). Conversely, a trend towards
a longer OS was observed in EC patients with high
miR375 expression, and patients with low miR-375 expression
had worse outcomes (Fig. 2b).We found that patients with
high expression levels of miR-21 were more likely to have
low expression levels of miR-375 (Table 4). We then
compared survival between (a) patients with high miR-375
Factor v2
0.443 0.506
0.145 0.704
7.632 0.022
3.366 0.067
0.336 0.845
1.720 0.190
1.004 0.229
0.126 0.722
6.610 0.037
1.711 0.191
0.964 0.618
0.877 0.349
Table 3 Prognostic factors according to the univariate analysis
Case OS rate (%)
1 year 3 years 5 years
expression and low miR-21 expression and patients with
either (b) high miR-21 expression or (c) low miR-375
expression. We found that the patients with both high
miR375 expression and low miR-21 expression had
significantly better outcomes (Fig. 2c).
In the univariate analysis, no significant relationships
were found between the miRNAs miR-21 and miR-375 and
patient characteristics including sex, age, Postoperative
metastasis and pathological type. Hence, only pathogenic
sites, TNM stage, the expression levels of miR-21 and
miR-375 were included in the multivariate analysis. The
multivariate analysis indicated that there was a significant
association between high expression levels of miR-21 and
poor OS in EC patients (HR 2.15, 95% CI 1.16–3.97). A
significant correlation was also found between a high level
of expression of miR-375 and improved OS in EC patients
(HR 0.47, 95% CI 0.26–0.87). In addition, TNM stage was
found to be correlated with OS (HR 2.36, 95% CI
1.19–4.70), but no correlation was found between
pathogenic sites and survival (HR 1.50, 95% CI 0.78–2.88)
(Table 5).
Forest plots evaluating the effect of miR-21
and miR-375
The prognostic value of using miR-21 and miR-375 was
then evaluated using Forest plot analyses. To investigate
the more general prognostic value of miR-21 and miR-375
in Asian populations, we performed Forest plots analyses
using data obtained from previously published studies. The
characteristics of the included studies were shown in
Tables 6 and 7. For miR-21, eight studies were selected for
the meta-analysis and 582 patients with esophageal cancer
totally. Six studies indicated that there was no significant
relationship between the expression level of miR-21 and
OS, whereas only two studies reported inverse correlations
between miR-21 expression levels and OS. However,
although these results were in disagreement, the Forest plot
analysis indicated that high expression levels of miR-21
significantly predicted a shortened OS with a pooled HR of
1.83 (95% CI 1.42–2.37) (Fig. 3a). For miR-375, six
studies with 318 esophageal cancer patients were selected
for inclusion in the Meta-analysis. Of those, three reported
that there was no significant correlation between miR-375
expression levels and prognoses, and the other three studies
indicated a significant association. The Forest plot analysis
resulted in a pooled HR of 0.56 (95% CI 0.43–0.73),
indicating that high miR-375 expression was significantly
correlated with increased survival (Fig. 3b).
The majority of the worldwide burden of EC occurs in the
‘‘Asian Esophageal Cancer Belt,’’ which extends from
northern Iran, east to China and north to Russia. In China,
the best-known region for high EC risk is the Taihang
Mountain which situated among the Hebei, Henan and
Shanxi Provinces. It covered about 90 million people. To
our knowledge, this is the first study to assess the potential
prognostic value of miRNAs in EC in an area associated
with a high-risk of EC. All of the patients in this study
came from Cixian in Hebei Province. Cixian and its
neighbour Linzhou in Henan Province are the areas with
the highest incidence of EC in China and worldwide. The
incidence of EC in Cixian is 176.9 per 100,000 in men and
108.8 per 100,000 in women (ASR, W).These rates are
much higher than the worldwide incidence (9.0 per
100,000 for men and 3.1 per 100,000 for women) [2].
The major risk factors known to contribute to the
development of EC include alcohol consumption, tobacco
use and nutritional deficiencies. The most common features
of high-risk areas are an under-developed economy, low
nutrient intake and limited health resources. Zou et al.
reported that zinc intake in high-risk areas was only 72 and
Fig. 2 a Overall survival in EC patients with high and low miR-21 expression. b Overall survival in EC patients with high and low miR-375
expression. c Overall survival in EC patients with both high miR-375 expression and low miR-21 expression
Table 4 Four-fold table for miR-21 and miR-375
62% of the recommended daily allowance (RDA) in the
spring and autumn, respectively, and this may be one of the
factors that contribute to the high-risk of EC in these areas
[10]. Epidemiological and clinical studies have implicated
dietary zinc deficiency in the pathogenesis of EC [11]. Zinc
is an essential trace element and a catalytic/structural
component that is used by many metalloenzymes and
transcription factors. Zinc availability is also important for
tumor growth and progression because zinc is a critical
component of many enzymes that are involved in hypoxia,
angiogenesis, cell proliferation, and cancer metastasis [12].
The Zn content is low in most foods, except for red meat
and seafood. People from areas with a high incidence of
EC who subsist mainly on a diet that does not include red
meat and seafood are likely to be ZD [10]. ZD increases the
risk of EC. In a rat model, chronic ZD induced an
inflammatory gene signature that fuelled EC development.
ZD was found to be a common phenomenon in areas with a
high incidence of EC, and an inflamed ZD oesophagus has
a distinct miRNA signature that resembles the miRNA
profile of human EC, with miR-21 being the most
upTable 5 Prognostic factors
according to the multivariate
analysis
regulated species [9]. Changes in miRNA levels may be
therefore associated with EC. The ZD-induced modulation
of the expression of specific miRNAs has been implicated
in the role of ZD in the pathogenesis of diseases [13].
Schetter et al. reported that miR-21 is the most
up-regulated oncomiRNA in human cancers and also the most
upregulated species in the ZD oesophagus [14]. ZD induces a
pro-oncogenic miRNA signature in the inflammatory
oesophagus, and the ZD oesophagus displays a distinct
miRNA signature that resembles the miRNA signature of
EC [13].
In this study, we first investigated the serum zinc levels
of patients and control group from high and low incidence
areas of EC. And the results showed that the serum zinc
levels of EC patients were lower than healthy people
whether in high incidence areas of EC or in low. The serum
zinc level of healthy people from high incidence areas of
EC was significantly lower than that from low incidence
areas could explain the reason of high incidence of EC
partly. The expression levels of miRNAs were investigated
and miR-21, miR-31, miR-93 and miR-375 were changed
when the Zn level was altered in EC cell lines. The
expression level of these miRNAs may therefore be subject
to alterations that are influence by ZD. The potential
prognostic value of these miRNAs was assessed in a cohort
study of 88 confirmed EC patients from areas with a high
incidence of EC in China. The results showed that only
miR-21 and miR-375 were related to prognoses.
There is consensus regarding the notion that miR-21 is
an oncogenic miRNA that is up-regulated in a variety of
human cancers and that over expression of miR-21 is
important in cancer development. MiR-21 is one of the
most frequently over expressed miRNAs in cancer, and it
exerts its anti-apoptotic effects by targeting the tumor
suppressors PDCD4 and PTEN in Certain types of cancer
[15–17], but there is no definite conclusion in EC. PDCD4
is one of the most frequently decreased proteins in EC, and
it is negatively regulated by miR-21. The down-regulation
of PDCD4 has been correlated with tumor stage and nodal
metastasis [16]. In addition, some reports have
demonstrated that the over expression of miR-21 is correlated
with poor clinical outcomes in EC. Recently, a large study
evaluated the potential prognostic value of miR-21 in
retrospective cohorts of 195 patients in a Western population,
and the expression level of miR-21 was significantly
RR (95% CI)
1.499 (0.780–2.880)
2.361 (1.187–4.696)
2.146 (1.159–3.972)
0.472 (0.257–0.868)
correlated with disease-specific survival in patients with
EC, while miR-21 was identified as an independent
prognostic biomarker [18]. A meta-analysis based on western
populations combined four studies, with three of the studies
reporting no significant association with survival whereas
only one showed a significant and negative correlation
between the expression level of miR-21 and survival.
A Forest plot analysis confirmed that this relationship had a
pooled HR of 1.56 (95% CI 1.18–2.07), indicating that a
high expression level of miR-21 significantly predicted a
poor prognosis [18].
In our study, the expression level of miR-21 was
significantly correlated with OS in patients with EC in both
the univariate analysis and the multivariate analysis. The
patients with high miR-21 expression levels had worse
outcomes, while better OS was observed in the patients
with low expression levels of miR-21. MiR-21 was
therefore identified as a biomarker of a poor prognosis. In
addition, we performed a meta-analysis that was based on
an Asian population [7, 15, 18–22].Of the included studies,
six found that there was no association between the
expression of miR-21 and prognosis, while only two
studied found that miR-21 expression levels were
significantly associated with survival. However, despite these
discordant results, the Forest plot analysis indicated that a
high expression level of miR-21 significantly predicted a
shortened OS, with a pooled HR of 1.83 (95% CI
1.42–2.37). MiR-21 was indeed found to be an independent
marker of a poor prognosis in EC, and this finding was in
agreement with our results. The HR for our results, which
were obtained by studying an area with a high incidence of
EC, was higher than the HR in the Forest plots that were
based on an analysis of a Western population or a general
Asian population. These results imply that miR-21 may be
a better prognostic marker in areas with a high incidence of
EC.
MiR-375 is known to play a role as a tumor suppressor,
and the expression of miR-375 is reduced in EC as a result
of promoter hypermethylation. It has been demonstrated
that miR-375 negatively regulates PDK1 and IGF1R and
that these effects contribute to the inhibition of tumor
proliferation and metastasis [23, 24]. In addition, Mathe
et al. reported that a low expression of miR-375 was
significantly correlated with a worse prognosis in EAC
patients with Barrett’s oesophagus [18]. Inconsistent data
8 2 5 6 1 4 1 0
R .1 .5 .4 .2 .5 .9 .4 .7
H 2 1 1 2 2 1 2 2
R R R R R R R R
M q q q q q q q q
s s s s
T e G G e e e G o
P Y N N Y Y Y N N
Fig. 3 a Forest plots evaluating the association between miR-21 expression and prognosis in ES. b Forest plots evaluating the association
between miR-375 expression and prognosis in ES
have been produced in previous studies regarding the
association between the expression of miR-375 and
prognoses. In a meta-analysis by Mette that was based on five
studies of Western populations, two of the studies reported
that there was no association between the expression of
miR-375 and prognosis, while three indicated that miR-375
expression levels were significantly associated with
survival. A high level of miR-375 was found to be
significantly correlated with increased survival in ESCC
(HR 0.62; 95% CI 0.49–0.79) [18].
Similarly, miR-375 was associated with patient
characteristics and prognoses in this study. Both the univariate
analysis and the multivariate analysis demonstrated that
there was a trend towards increased survival in patients
with high levels of miR-375 (HR 0.47, 95% CI 0.26–0.87).
A meta-analysis based on data from studies of Asian
populations was performed, and we found that a low level
of miR-375 was significantly associated with a worse
prognosis (HR 0.56; 95% CI 0.43–0.73) [7, 18, 19, 24–26].
This finding is in disagreement with studies of general
Asian populations or Western populations. miR-375 is
thought to be a prognostic biomarker that acts as an
antionco miR. The HR in an area with a high incidence of EC
was lower than the HR in the Forest plot analyses, whether
they were based on a general Asian population or a
Western population. These result simply that miR-375 might be
a better prognostic marker in areas with a high incidence of
EC.
In addition, we found it interesting that the patients with
high levels of miR-21 were more likely to have low
expression levels of miR-375. To further verify the
prognostic value of miR-21 and miR-375, survival was
analyzed in patients with both high miR-375 expression and
low miR-21 expression, and the results were compared to
survival in patients with either high miR-21 expression or
low miR-375 expression. We found that the patients with
both high miR-375 expression and low miR-21 expression
had significantly better outcomes. These results support the
notion that combining analyses of miR-21 and miR-375
levels may have an improved effect in comparison to
analyses of either marker alone.
The clinical rational for using prognostic biomarkers,
such as miRNAs, is their potential to predict clinical
outcomes. This enables clinicians to identify high-risk patients
who might benefit from more aggressive treatment
strategies and identify low-risk patients who might be safely
treated with less intensive treatment regiments. We found
that miR-21 and miR-375 are prognostic biomarkers in
areas with a high incidence of EC and that they may be
used to better effect when combined.
Acknowledgements This study was supported by grants from the
National Natural Scientific Foundation of China (81272682) and the
National Natural Scientific Foundation of Hebei Province
(C2011206058).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
1. GLOBOCAN: estimated cancer incidence, mortality and prevalence worldwide in 2012 . http://globocan.iarc.fr/Pages/fact sheetscancer.aspx
2. He J , Chen W ( 2012 ) Chinese cancer registry annual report , 2012 . Press of Military Medical Sciences, Beijing, pp 160 - 161
3. Zeng H , Zheng R , Guo Y , Zhang S , Zou X , Wang N , Zhang L , Tang J , Chen J , Wei K et al ( 2015 ) Cancer survival in China, 2003-2005: a population-based study . Int J Cancer 136 : 1921 - 1930
4. Zhu YH , Fu L , Chen L , Qin YR , Liu H , Xie F , Zeng T , Dong SS , Li J , Li Y et al ( 2013 ) Downregulation of the novel tumor suppressor DIRAS1 predicts poor prognosis in esophageal squamous cell carcinoma . Cancer Res 73 : 2298 - 2309
5. Esquela-Kerscher A , Slack FJ ( 2006 ) Oncomirs-microRNAs with a role in cancer . Nat Rev Cancer 6 : 259 - 269
6. Calin GA , Sevignanic C , Dumitru CD et al ( 2004 ) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers . Proc Natl Acad Sci USA 101 ( 9 ): 2999 - 3004
7. Li BX , Yu Q , Shi ZL , Li P , Fu S ( 2015 ) Circulating microRNAs in esophageal squamous cell carcinoma: association with locoregional staging and survival . Int J Clin Exp Med 8 : 7241 - 7250
8. Abnet CC , Lai B , Qiao YL , Vogt S , Luo XM , Taylor PR , Dong ZW , Mark SD , Dawsey SM ( 2005 ) Zinc concentration in esophageal biopsy specimens measured by x-ray fluorescence and esophageal cancer risk . J Natl Cancer Inst 97 : 301 - 306
9. Fong LY , Taccioli C , Jing R , Smalley KJ , Alder H , Jiang Y , Fadda P , Farber JL , Croce CM ( 2016 ) MicroRNA dysregulation and esophageal cancer development depend on the extent of zinc dietary deficiency . Oncotarget 7 : 10723 - 10738
10. Zou XN , Taylor PR , Mark SD , Chao A , Wang W , Dawsey SM , Wu YP , Qiao YL , Zheng SF ( 2002 ) Seasonal variation of food consumption and selected nutrient intake in Linxian, a high risk area for esophageal cancer in China . Int J Vitam Nutr Res 72 : 375 - 382
11. Hashemian M , Poustchi H , Abnet CC , Boffetta P , Dawsey SM , Brennan PJ , Pharoah P , Etemadi A , Kamangar F , Sharafkhah M et al ( 2015 ) Dietary intake of minerals and risk of esophageal squamous cell carcinoma: results from the Golestan Cohort Study . Am J Clin Nutr 102 : 102 - 108
12. Gaither LA , Eide DJ ( 2001 ) Eukaryotic zinc transporters and their regulation . Biometals 14 : 251 - 270
13. Alder H , Taccioli C , Chen H , Jiang Y , Smalley KJ , Fadda P , Ozer HG , Huebner K , Farber JL , Croce CM et al ( 2012 ) Dysregulation of miR-31 and miR-21 induced by zinc deficiency promotes esophageal cancer . Carcinogenesis 33 : 1736 - 1744
14. Schetter AJ , Heegaard NH , Harris CC ( 2010 ) Inflammation and cancer: interweaving microRNA, free radical, cytokine and p53 pathways . Carcinogenesis 31 : 37 - 49
15. Zhao Y , Schetter AJ , Yang GB , Nguyen G , Mathe´ EA , Li P , Cai H , Yu L , Liu F , Hang D et al ( 2013 ) microRNA and inflammatory gene expression as prognostic marker for overall survival in esophageal squamous cell carcinoma . Int J Cancer 132 : 2901 - 2909
16. Isanejad A , Alizadeh AM , Amani Shalamzari S , Khodayari H , Khodayari S , Khori V , Khojastehnjad N ( 2016 ) MicroRNA-206, let-7a and microRNA-21 pathways involved in the anti-angiogenesis effects of the interval exercise training and hormone therapy in breast cancer . Life Sci 151 : 30 - 40
17. Peralta-Zaragoza O , Deas J , Meneses-Acosta A , De la O- Go´mez F , Ferna´ndez-Tilapa G , Go´mez-Cero´n C , Ben´ıtez-Boijseauneau O , Burguete-Garc ´ıa A, K Torres-Poveda , Bermu´dez-Morales VH et al (2016) Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells . BMC Cancer 16 : 215
18. Winther M , Alsner J , Tramm T , Baeksgaard L , Holtved E , Nordsmark M ( 2015 ) Evaluation of miR-21 and miR-375 as prognostic biomarkers in esophageal cancer . Acta Oncol 54 : 1582 - 1591
19. Mathe ´ EA, Nguyen GH , Bowman ED , Zhao Y , Budhu A , Schetter AJ , Braun R , Reimers M , Kumamoto K , Hughes D et al ( 2009 ) MicroRNA expression in squamous cell carcinoma and adenocarcinoma of the esophagus: associations with survival . Clin Cancer Res 15 : 6192 - 6200
20. Komatsu S , Ichikawa D , Takeshita H , Konishi H , Nagata H , Hirajima S , Kawaguchi T , Arita T , Shiozaki A , Fujiwara H et al ( 2012 ) Prognostic impact of circulating miR-21 and miR-375 in plasma of patients with esophageal squamous cell carcinoma . Expert Opin Biol Ther 12 (Suppl 1): S53 - S59
21. Hamano R , Miyata H , Yamasaki M , Kurokawa Y , Hara J , Moon JH , Nakajima K , Takiguchi S , Fujiwara Y , Mori M et al ( 2011 ) Overexpression of miR-200c induces chemoresistance in esophageal cancers mediated through activation of the Akt signaling pathway . Clin Cancer Res 17 : 3029 - 3038
22. Xiang Z , Yan B ( 2014 ) MicroRNA-21 expression in esophageal cancer and its clinical significance . Cancer Res Prev Treat 41 : 902 - 905
23. Li X , Lin R , Li J ( 2011 ) Epigenetic silencing of microRNA-375 regulates PDK1 expression in esophageal cancer . Dig Dis Sci 56 : 2849 - 2856
24. Kong KL , Kwong DL , Chan TH , Law SY , Chen L , Li Y , Qin YR , Guan XY ( 2012 ) MicroRNA-375 inhibits tumour growth and metastasis in oesophageal squamous cell carcinoma through repressing insulin-like growth factor 1 receptor . Gut 61 : 33 - 42
25. Li J , Li X , Li Y , Yang H , Wang L , Qin Y , Liu H , Fu L , Guan XY ( 2013 ) Cell-specific detection of miR-375 downregulation for predicting the prognosis of esophageal squamous cell carcinoma by miRNA in situ hybridization . PLoS ONE 8:e53582
26. Wu C , Li M , Hu C , Duan H ( 2014 ) Clinical significance of serum miR-223, miR-25 and miR-375 in patients with esophageal squamous cell carcinoma . Mol Biol Rep 41 : 1257 - 1266