1
DNMT1-microRNA126 epigenetic circuit contributes to esophageal
squamous cell carcinoma growth via ADAM9-EGFR-AKT signaling
Ronghua Liu a, b, #, Jie Gu c, #, Pei Jiang a, b, Yijie Zheng a, b, Xiaoming Liu a, b, Xuechao
Jiang a, b , Enyu Huang a, b , Shudao Xiong a, d ,Fengkai Xu c, Guangwei Liu a, b, Di Gec,*,
Yiwei Chua, b, *
a Department of Immunology, Key Laboratory of Medical Molecular
Virology of MOE/MOH, School of Basic Medical Sciences, Fudan University,
Shanghai,200032, People's Republic of China.
b Biotherapy Research Center, Fudan University, Shanghai 200032, China.
c Department of Thoracic Surgery, The Affiliated Zhongshan Hospital of Fudan University,
Shanghai, 200032, People's Republic of China.
d Department of Hematology/Oncology, The Second Hospital of Anhui Medical University,
Hefei, Anhui Province, People's Republic of China.
# These authors contributed equally to this paper.
*Correspondence:
Yiwei Chu, M.D, Ph.D, Department of Immunology and Key Laboratory of Molecular,
Medicine of Ministry of Education, Shanghai Medical College, Fudan University, No.138,
Yi Xue Yuan Rd., mail box 226, Shanghai, 200032, P.R.China, Tel. /fax: +86 21 54237324,
E-mail address: [email protected]; or Di Ge, M.D, Department of Thoracic Surgery,
Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, P.R.China,
Tel. /fax: +86 21 64041990-2559, E-mail address. [email protected].
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RUNNING TITLE: DNMT1-microRNA126 epigenetic circuit regulates ESCC growth
Key Words: epigenetics; microRNA; DNA methylation; prognosis
Number of words in text: 4870;
Supplementary Tables: 6; Supplementary Figures: 6.
Disclosure of Potential Conflicts of Interest
The authors declare no conflicts of interest.
Translational Relevance
Esophageal squamous cell carcinoma growth (ESCC) is one of the most aggressive
malignancies of the gastrointestinal tract. Previous studies have described complicated
genetic and epigenetic alternations involved in ESCC progression, but the underlying
mechanisms have not been fully elucidated. Recent evidences show that microRNAs
(miRNAs) are involved in and are controlled by epigenetic regulation, and thereby form a
reciprocal regulatory circuit in many systems. In this study, using NGS-based miRNA
profiling, we found the downregulation of miR-126 in ESCC and proposed a
“DNMT1-miR-126 epigenetic circuit” involved in ESCC progression. Moreover, miR-126
acted as a novel tumor suppressor that effectively suppresses ESCC cell proliferation and
migration by repressing ADAM9-EGFR-AKT signaling transduction and as an
independent prognostic indicator for ESCC patients. Taking together, our funding of
“DNMT1-miR-126 epigenetic circuit” suggests another insight into pathological
mechanisms underlying ESCC initiation and progression, implying its clinical significance
in developing targets for ESCC prediction and therapy.
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Abstract
Purpose: MicroRNAs (miRNAs) are involved in and are controlled by epigenetic
regulation, and thereby form a reciprocal regulatory circuit. Using next-generation
sequencing (NGS)-based miRNA profiling, this study aimed to discover esophageal
squamous cell carcinoma (ESCC)-specific miRNAs and miRNA-related epigenetic
modulations.
Experimental Design: NGS-based miRNA profiles were generated for 4 pairs of ESCC
tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA
expression and its correlation with prognosis. miRNA-related DNA methylations were
identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1
(DNMT1) was investigated using RNA interference. miRNA targets were screened by
mRNA sequencing, and functional validation was performed in vitro and in vivo.
Results: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in
ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its
downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was
due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly
upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly,
DNMT1 was suppressed by overexpression of miR-126, indicating the existence a
regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic
expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and
migration by inhibiting epidermal growth factor receptor-AKT signaling.
Conclusions: Our results indicate that miR-126 is a potential prognostic indicator for
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ESCC and suggest that a novel “DNMT1-miR-126 epigenetic circuit” is involved in ESCC
progression. Consequently, miR-126-based epigenetic modulations may provide a basic
rationale for new approaches to anti-tumor therapeutics.
Introduction
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive
malignancies of the gastrointestinal tract(1). Despite improvements in diagnostic
approaches and the introduction of adjuvant chemotherapy, the 5-year survival rate in
patients with esophageal cancer remains less than 30%(2). Previous studies have
described complicated genetic and epigenetic alternations involved in ESCC progression
(3, 4), but the underlying mechanisms have not been fully elucidated. Therefore, in order
to develop better approaches for ESCC diagnosis and therapy, a comprehensive
understanding of the molecular variables that affect ESCC pathways is needed.
MicroRNAs (miRNAs) have emerged as key players in human tumors; their
dysregulation affects cell proliferation(5), differentiation(6), invasion(7), and the
epithelial-mesenchymal transition (EMT)(8). miRNA expression profiles have been
developed for use as prognostic/predictive biomarkers and been used to identify the
molecular signatures and underlying functional mechanisms of various cancers. Previous
miRNA profiling studies were conducted using miRNA expression microarray platforms (9,
10). However, in recent decades, the emergence of next-generation sequencing (NGS)
platforms has revolutionized genomic studies. In comparison with microarray, NGS-based
sequencing has advantages for the analysis of widely expressed miRNAs, the
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identification of miRNA sequence variations, and the discovery of novel miRNAs(11, 12).
Although, microarray-based miRNA profiles of ESCC have been described (13), to our
knowledge, no studies have used an NGS-based platform to identify ESCC-specific
miRNAs. Furthermore, the pathological mechanisms underlying their dysregulation
remain incompletely understood.
Epigenetic modulations, including DNA methylation and histone acetylation, play a
critical role in various diseases by regulating gene expression without changing the DNA
sequence(14). Recent evidence indicates that miRNAs integrate into the epigenetic
machinery via the transcriptional regulation of epigenetic factors, creating an
“epigenetic-miRNA feedback loop” (15, 16). In this study, we propose that a
“DNMT1-miR-126 epigenetic circuit” is involved in ESCC progression. MiR-126 was
silenced by promoter hypermethylation of its host gene in ESCC tissues, while DNMT1
was aberrantly overexpressed. Silencing of DNMT1 increased miR-126 expression by
reducing the hypermethylation, while miR-126 suppressed DNMT1 expression via a
negative feedback process. MiR-126 was also identified as a novel tumor suppressor and
a potential prognostic indicator in ESCC. Overexpression of miR-126 repressed ESCC
cell proliferation and migration by inhibiting the activation of the ADAM9/EGFR/AKT
pathway. Understanding the epigenetic-miRNA network in ESCC progression and
modulating miRNA-related epigenetic dynamics hold promise for ESCC targeted therapy.
Materials and Methods
Samples and cells
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ESCC specimens embedded in paraffin were obtained from 185 patients who underwent
esophagectomy at Zhongshan Hospital (Shanghai, People's Republic of China) in 2007
with informed consent, and clinicopathological characteristics of patients were listed in the
supplementary table S1. Charlson comorbidity index (CCI) was used for the analysis of
comorbidities(17). Each paraffin blocks was well formalin-fixed, paraffin-embedded and
without necrosis and hemorrhage. Follow-up was conducted every six mouths, and ended
in December 2012, with a median follow-up of 32 months. The clinical endpoint was the
survival state of patient at the last follow-up. Disease-specific survival was defined as the
interval between surgery and death of ESCC. The data were censored for patients who
died of causes other than ESCC or survived at the last follow-up. Another 30 pairs of
frozen human primary human ESCC tissues (ECs) and matched adjacent non-cancerous
tissues (NMs) were obtained from 30 patients at Zhongshan Hospital in 2012. Tumor
stage was determined according to 7th edition of the Union for International Cancer
Control-American Joint Committee on Cancer tumor, node, metastasis staging system
(18). Approval for the study was obtained from the Research Ethics Committee of
Zhongshan Hospital.
ESCC cell line ECa-109 was obtained from the Institute of Biochemistry and Cell
Biology of the Chinese Academy of Science (TCHu 69, Shanghai, China), and ESCC cell
line KYSE-510 (JCRB1436, National Institute for Biomedical Innovation, Japan) and
normal human esophageal epithelial cell line HEEpiC (HEEC) (#2720, ScienCell
Research Laboratories, USA) was supplied by Dr. Lu (19), which were authenticated
using short tandem repeat (STR) profiling and used in the 6 month after receipt in this
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research. All the cells were cultured in RPMI-1640 supplemented with 10% FBS, 2 μM
glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin sulfates.
NGS-based miRNA profiling
Total RNA from 4 pairs of ECs and matched NMs were isolated using a mirVana miRNA
Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. After
PAGE purification, small RNA molecules were amplified and sequenced on an Illumina
platform according to the manufacturer’s instructions (20).
Tissue microarray and in situ hybridization (ISH)
Tissue microarrays were constructed as described in a previous study(21). Digoxin
(DIG)-labeled oligonucleotide probes complementary to hsa-miR-126 were designed as
previously described and purchased from ExonBIO (Guangzhou, China)(22). The probe
sequence was 5′-UCGUACCGUGAGUAAUAAUGCG-3′. The slides were hybridized with
DIG-conjugated hsa-miR-126 probes overnight at 42°C and stained using an Enhanced
Sensitive ISH Detection Kit II (AP) with phosphatase-conjugated anti-DIG antibody
(BOSTER, China) according to the manufacturer’s instructions.
Statistical analysis
Data are presented as the mean ± SEM of replicate experiments (n > 3). Results were
analyzed using SPSS 16.0 software (SPSS, Chicago, IL, USA) and PRISM 5.0
(GraphPad Software Inc., San Diego, CA, USA). Student’s unpaired t-test or unpaired
t-test with Welch's correction was used to analyze intergroup differences for 2 groups,
ANOVA was used to analyze more than 2 groups, and Pearson’s correlation coefficient
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was used to analyze the correlation between groups. The cumulative survival time was
calculated using the Kaplan-Meier method and analyzed using the log-rank test.
Univariate and multivariate analyses were based on the Cox proportional hazards
regression model. P values < 0.05 were considered statistically significant.
Results
NGS-based discovery of miR-126 and validation of its downregulation in ESCC
To identify ESCC-specific miRNA profiles, NGS-based miRNA profiling was performed on
4 pairs of frozen ECs and adjacent NMs. When ECs and NMs were compared, 78
miRNAs were differentially expressed, of which 44 were downregulated and 34 were
upregulated in ECs (P < 0.05 with fold-change > 3) (Fig. 1A and Supplementary Table
S2). Among the differentially regulated miRNAs, the downregulation of miR-126-3p in ECs
exhibited the strongest statistical significance. For validation, qPCR was used to assess
the expression of miR-126 in 30 pairs of frozen ECs and matched NMs, as well as in
ESCC cell lines (ECa-109 and KYSE-510) and HEECs, normal human esophageal
epithelial cells. The results showed that miR-126 expression was also downregulated in
ESCC tissues (P < 0.001) (Fig. 1B) and cell lines (P < 0.001) (Fig. 1C), when compared
with expression in NMs and HEECs, respectively.
Downregulation of miR-126 correlates with poor prognosis in ESCC patients
To address the clinical significance of miR-126 in ESCC, ISH was performed in 185 ESCC
specimens. Patients with negative miR-126 expression exhibited lower 5-year
disease-specific survival rate than that with positive miR-126 expression (P = 0.014,
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Figure 1D). In multivariate analysis, CCI, tumor stage and miR-126 expression were
identified as independent prognostic factors in patients’ disease-specific survival
(Supplementary Table S3). These results suggest that downregulation of miR-126 is an
independent indicator of prognosis in ESCC.
Hypermethylation of the Egfl7 promoter induces miR-126 downregulation in EC
Given that the miR-126 gene is located in an intron of Egfl7 and its expression is
reportedly upregulated in cancer cells treated with inhibitors of DNA methylation(23), we
analyzed the sequence of Egfl7 and found that a large CpG island was present in the
promoter region (Fig. 2A). To understand the correlation between methylation of the Egfl7
promoter and the downregulation of miR-126, we analyzed CpG island methylation in ECs
and matched NMs. The results showed that Egfl7 promoter methylation was higher in ECs
than in NMs (Fig. 2B). Samples with higher methylation exhibited lower miR-126
expression (Fig. 2C). To determine the effect of methylation of the Egfl7 promoter on the
expression of miR-126, genomic DNA was extracted from ECa-109 and KYSE-510 cells
treated with 5-azadC or vehicle. After sodium-bisulfite modification, MSP was carried out
using the specific primers for the unmethylated (U) or methylated (M) Egf7 promoter
sequence. As a Result, compared with vehicle treated cells in which the Egf7 promoter
was hypermethylated, cells treated with the demethylating agent 5-aza-dC to induce
demethylation of the Egfl7 promoter showed more unmethylated sequences and an
increase in miR-126 (Fig. 2D). These results indicate that hypermethylation of the Egfl7
promoter directly downregulates miR-126 in ESCC.
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DNMT1-miR-126 regulatory circuit contributes to Egfl7 promoter hypermethylation
DNA methyltransferases (DNMTs) are critical for gene-specific methylation and the
consequent transcriptional silencing of cancer-related genes(24). In this study, we found
that DNMT1 expression was dramatically increased in ESCC tumor tissues and cell lines
(Fig. 3A, Supplementary Fig. S1A). Furthermore, correlation analysis showed that the
expression of DNMT1 inversely correlated with that of endogenous miR-126 (Fig. 3A). To
investigate the role of DNMT1 in the regulation of miR-126 expression, we silenced
DNMT1 expression in cells using siRNA. DNMT1 knockdown reduced the
hypermethylation of the miR-126 host gene (Fig. 3B), leading to miR-126 upregulation
(Fig. 3C). Interestingly, overexpression of miR-126 suppressed DNMT1 expression in
both KYSE-510 and ECa-109 cells (Fig. 3D). Collectively, overexpression of DNMT1
resulted in the hypermethylation and downregulation of miR-126, while overexpression of
miR-126 suppressed DNMT1 expression. Our findings indicate the involvement of a novel
DNMT1-miR-126 regulatory loop in ESCC.
Overexpression of miR-126 suppresses ESCC cell proliferation and migration
Given the low expression of miR-126 in ESCC, we further evaluated the biological role
played by miR-126 in ESCC cell lines. miR-126 mimics were transfected into ECa-109
and KYSE-510 cells to upregulate miR-126 expression (Supplementary Fig. S2A).
Overexpression of miR-126 markedly suppressed cell proliferation (Fig. 4A) and the rate
of colony formation (Fig. 4B) in ECa-109 and KYSE-510 cells when compared to the
negative control. Further analysis showed that miR-126 mainly promoted cell apoptosis
(Supplementary Fig. S2B), but without a marked effect on the cell cycle (Supplementary
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Fig. S2C). In addition, overexpression of miR-126 inhibited EC cell migration in vitro (Fig.
4C, Supplemetary Fig. S3A).
To confirm the suppressive role of miR-126 in ESCC, we next investigated the effect
of miR-126 on ESCC tumor growth in vivo. ECa-109 and KYSE-510 cells transfected with
either miR-126 or miR-NC (control group) were implanted subcutaneously into the
bilateral posterior flank of nude mice. Compared to tumor growth in the control group,
tumor growth in the miR-126-transfected group was markedly suppressed, as reflected by
the smaller tumor sizes (Fig. 4D, Supplemetary Fig. S3B). Collectively, these results
indicate that miR-126 functions as a tumor suppressor by inhibiting ESCC cell growth and
migration.
The ADAM9/EGFR/AKT signaling directly mediates the suppressive role of miR-126
To investigate the targets and downstream signaling pathway of miR-126, we compared
mRNA profiles of ECa-109 cells transfected with miR-126 (126-EC cells) or miR-NC
(NC-EC cells) mimics using NGS-based mRNA sequencing. In 126-EC cells, 31 genes
were significantly downregulated relative to the levels in NC-EC cells (Supplementary
Table S4), including ADAM9. Consistently, in bioinformatics analysis, ADAM9, a putative
target gene of miR-126, was identified with 4 prediction algorithms (PicTar, TargetScan,
miRanda, and miRDB) (Supplementary Table S4). Therefore, ADAM9 was selected for
further validation and functional analyses. Results from qPCR, western blot, and
immunohistochemistry (IHC) showed that ADAM9 was overexpressed in ECs and ESCC
cell lines and that its mRNA levels inversely correlated with miR-126 levels in ECs (Fig.
5A-B, Supplementary Fig. S4A). This inverse correlation strongly suggested the
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involvement of ADAM9 in the miR-126-related network. Bioinformatics analysis showed
that the binding sites in the 3′-UTR of ADAM9 and miR-126 are highly conserved (Fig. 5C).
We constructed the ADAM9-3′-UTR reporter plasmid and performed luciferase reporter
assays in cells transfected with the 3′-UTR plasmid. As expected, miR-126 repressed
luciferase activity in cells expressing ADAM9-3′-UTR, indicating a direct interaction
between miR-126 and ADAM9 in ESCC cells (Fig. 5C). Furthermore, a decrease in
ADAM9 was detected in miR-126-overexpressing ESCC cells (P < 0.05) (Fig. 5D). To
address the direct involvement of ADAM9 in miR-126-regulated tumor suppression,
ECa-109, KYSE-510, and KYSE-510 cells were transfected with siRNAs against ADAM9
mRNA; si-1 (si-ADAM9) was selected for further study because of its efficient interference
(Supplementary Fig. S4B). EC cell proliferation was inhibited in
ADAM9-siRNA-transfected ECa-109 and KYSE-510 cells and the cell migration rate was
lower than in si-Ctrl-transfected cells (Fig. 6A). Moreover, after silencing DNMT1, the
expression of ADAM9 in protein level was also reduced, with decreasing cell viability and
migration (Fig. 5D, Supplementary Fig. S1B–C). These data indicate that ADAM9 is the
target of miR-126 and is regulated by the “DNMT1-miR-126 circuit” in ESCC.
ADAM9 activates EGFR by cleaving the transmembrane pro-heparin-binding
epidermal growth factor-like growth factor (pro-HB-EGF) precursor to yield HB-EGF, a
soluble EGFR ligand. HB-EGF binds and activates intracellular signaling cascades
downstream of EGFR, such as the AKT pathway, and thereby promotes the growth of
normal and neoplastic cells (25, 26). In our study, we found that the EGFR/AKT pathway
was involved in the suppressive action of miR-126/ADAM9 in ESCC. Both overexpression
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of miR-126 and blockage of ADAM9 reduced levels of p-EGFR and p-AKT (Fig. 6B), cell
proliferation, and cell migration decreased (Fig. 6C). These results indicate that the
ADAM9/EGFR/AKT pathway directly mediates the suppressive action of miR-126 in
ESCC (Fig. 6D).
Discussion
The ”epigenetic-miRNA loop,” wherein miRNAs regulate or are regulated by epigenetic
factors, was proposed by Pagano et al(27). They suggested that epigenetic-miRNA loops
contributed to hematopoiesis by orchestrating gene expression in hematopoietic cells. In
this study, we identified the DNMT1-miR-126 epigenetic loop in ESCC and investigated its
contribution to ESCC development via modulation of the ADAM9/EGFR/AKT pathway.
Dysregulation of miRNAs in diseases is frequently due to epigenetic changes in the
miRNA genome(28, 29). Previous studies of miRNA genomic sequences have indicated
that promoter hypermethylation of miRNAs occurs frequently as a result of the aberrant
expression of DNMTs, histone deacetylases, and methyl-CpG-binding proteins (29-31). In
the present study, we found that miR-126 was downregulated in ECs and that its
downregulation was induced by promoter hypermethylation of its host gene.
Dysregulation of miR-126 has also been observed in other tumors, such as colon
cancer(32) and lung cancer (33), but little is known about its function in ESCC. This is the
first report to show that miR-126 is downregulated and acts as a new tumor suppressor in
ESCC. Moreover, we found that downregulation of miR-126 is a potential prognostic
indicator for ESCC. However, the results was different from the prior report that
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miRNA-126 failed to show a relation to outcome of patients with esophageal
adenocarcinoma (34). Such kind of conflict might due to the distinct biology and clinical
features of esophageal squamous carcinoma in our study and esophageal
adenocarcinoma in the previous study. The differences in races (Asian vs. American)
might also contribute to it.
Although evidence from Zhang et al. indicated that the downregulation of miR-126 in
breast cancer positively correlated with hypermethylation of the Egfl7 T2 promoter (35),
the underlying molecular mechanism was not elucidated. Notably, our results demonstrate
that DNMT1 is overexpressed in primary ECs and cell lines and that DNMT1 responsible
for the hypermethylation of the Egfl7 promoter, leading to miR-126 downregulation.
DNMT1, a member of the DNMT family (DNMT1, DNMT3A, and DNMT3B), is
overexpressed in tumors and is essential for maintaining promoter hypermethylation of
tumor suppressive genes, leading to their silencing (36). In breast cancer, overexpressed
DNMT1 induces miR-148a/152 silencing by maintaining the hypermethylation of their
promoters and contributes to tumor transformation and angiogenesis (37). In ESCC, we
demonstrate that overexpressed DNMT1 was responsible for the promoter
hypermethylation of the miR-126 host gene, thereby downregulating miR-126 and
inhibiting ESCC growth and cell migration. Interestingly, in a feedback mechanism,
DNMT1 was suppressed by miR-126 overexpression in ESCC cells, indicating the
presence of a novel “DNMT1-miR-126 epigenetic circuit.” We also showed that DNMT1
was the direct target of miR-126 (Supplementary Fig. S5). Regulation of DNMT1 by
miR-126 has also been described in CD4+ T cells from systemic lupus erythematosus
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patients (37). Similar to the “DNMT1-miR-126 epigenetic circuit” in ESCC, a negative
feedback regulatory loop between miR-148/152 and DNMT1 has been reported in breast
cancer (38). Thus, epigenetic-miRNA loops are widely present in various tumors. Given
that both miRNA and epigenetic modulations strongly regulate the expression of various
disease-associated genes(14), controlling the dynamic balance of epigenetic-miRNA
loops might be of great clinical significance.
The expression of ADAM9 has been reported to correlate with cell adherence and
migration (25, 33, 39, 40). This study found that ADAM9 functioned as a direct target of
miR-126 and contributed to miR-126 repressing cell migration in ESCC. Consistently, the
targeting of miR-126 to ADAM9 and the role of ADAM9 in cell migration was also reported
in a previous study by S. Hamada et al (39). A similar approach with our study using
restoration of miR-126 and silencing of ADAM9 showed inhibition of the invasive growth.
In order to systematically investigate the target of miR-126 in ESCC, RNA sequencing
was applied in the present study to examine the differential gene expression in
miR-126-overexpressed ESCC EC-109 cells vs. control cells. ADAM9 was showed to be a
functional target of miR-126, while other potential targets are under exploration. Therefore,
apart from pancreatic cancer in S. Hamada’s study, this study also identified the
interaction between miR-126 and ADAM9 in ESCC. In addition, we investigated the
down-stream pathway and found that ADAM9 was critical for promoting cell proliferation in
ESCC by targeting EGFR-AKT signaling. ADAM9 cleaved the pro-HB-EGF precursor to
yield soluble HB-EGF, which binds to EGFR, thereby activating downstream signal
transduction (26, 41). The EGFR/AKT pathway is important for cell growth and migration
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(42, 43). Our results showed that blocking ADAM9 inhibits EGFR/AKT activation by
suppressing phosphorylation, but has no effect on ERK phosphorylation. We also found
that the expression of phosphoinositide-3-kinase, regulatory subunit 2 (beta) was
downregulated upon miR-126 overexpression (Supplementary Table S4 and
Supplementary Fig. S6A). However, its expression in ECs and in NMs did not differ (P >
0.05), and its inverse correlation with miR-126 expression was poor (R=-0.1791,
P=0.5255) (Supplementary Fig. S6B-C). Other growth-related targets of miR-126
identified in other tumors, such as Crk(44) and IRS(45), were not downregulated in
miR-126-overexpressing ECa-109 cells (Supplementary Fig. S6A), implying they are not
key targets of miR-126 in ESCC growth.
With accumulating evidences demonstrating the importance of miRNAs in cancer
biology, miRNA based cancer therapy emerges as a hot issue and also a big challenge in
the current miRNA research. To be excited, MRX34, as the first microRNA-based therapy
for cancer, is entering the phase 1 trial(46). In the present study, our primary data showed
that the exogenous miR-126 mimics suppressed ESCC cell growth, which implies its
potential therapeutic significance in ESCC using a miRNAs’ “replacement” strategy.
However, miR-126 based therapy for ESCC in practice still needs to be further explored,
considering some challenges, especially technical limitations, such as how to improve its
accumulation in the target tissues and balance the safety and efficiency. In general, there
is a long way ahead to realize the translation of fundamental research of miR-126 to
clinical applications in ESCC.
In summary, this study proposes a “DNMT1-miR-126 epigenetic circuit” in ESCC
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and sheds light on its importance for ESCC progression via regulation of
ADAM9-EGFR-AKT signaling. Altering critical gene expression and signal transduction by
modulating miR-126 related epigenetic dynamics might be of clinical significance in ESCC
prediction and therapy.
Grant Support
This work was supported by the National Science Foundation of China (81273215,
81372313, 81401876 and 81272259), the Major Research Plan of the National Natural
Science Foundation of China (91229110), the National Basic Research Program of China
(973 Programs, 2011CB910404) and the Specialized Research Fund for the Doctoral
Program of Higher Education (20120071110046).
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Figure legends
Figure 1. NGS-based discovery of miR-126 and validation of its downregulation in
ESCC. (A) Heatmap and sample clustering analysis of the miRNAs differentially
expressed in ECs and NMs, and the Pearson correlation coefficient are shown. The
expression of miR-126 detected by RNA-Seq was confirmed by qPCR. (B) Levels of
miRNA-126 in 30 matched ECs and NMs were detected using qPCR and normalized to
U6 levels. The percentage of ESCC samples with upregulated, downregulated, and
unchanged miR-126 expression was analyzed. The qPCR results are shown as the mean
± SD (***P < 0.001). (C) Relative levels of miR-126 were detected in ECa-109, KYSE-510,
and HEECs by qPCR and normalized to U6 levels. The mean ± SD was determined from
3 replicates (***P < 0.001). (D) ISH was used to detect the presence of miR-126 in ECs.
Kaplan-Meier analysis showed that negative miR-126 expression was associated with
poor disease-specific survival in ECs (P = 0.014, log-rank test).
Figure 2. Hypermethylation of the Egfl7 promoter induces miR-126 downregulation
in ESCC. (A) The location of the miR-126 gene (MIR 126) in Egfl7 was analyzed. The
CpG island consisting of 12 CpG sites was used for bisulfite genomic sequencing (BSP)
and methylation-specific PCR (MSP) analyses. (B) BSP was used to determine the
methylation ratio at the Egfl7 promoter in ECs (n = 4) and matched NMs (n = 4). Black
circles and open circles represent methylated (M) and unmethylated (U) CpG
dinucleotides respectively. Data shown are the mean ± SD (**P < 0.01). (C) The
correlation between miR-126 levels (black bars) and Egfl7 promoter methylation levels
(red lines) was analyzed in the 4 pairs of esophageal samples. (D) Egfl7 promoter
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methylation levels and miR-126 expression were determined by MSP and qPCR
respectively both in ECa-109 and KYSE-510 cells treated with 5-aza-dC (5 µM) or vehicle.
MSP was carried out using the specific primers for the unmethylated (U) or methylated (M)
Egf7 promoter sequence. The mean ± SD was determined from 3 replicates (***P <
0.001).
Figure 3. DNMT1-miR-126 regulatory circuit contributes to Egfl7 promoter
hypermethylation. (A) DNMT1 RNA levels in 30 matched ECs and their NMs were
detected using qPCR and normalized to GAPDH expression. Data shown are the mean ±
SD values (**P < 0.01). DNMT1 RNA levels in cancer tissues negatively correlated with
miR-126 levels (P = 0.0074; R = -0.4787). DNMT1 protein levels were measured in 6
pair-matched ESCC samples and in cell lines. (B) ECa-109 and KYSE-510 cells were
transfected with control RNA (si-Ctrl, 100 nM) or DNMT1 siRNA (si-DNMT1, 100 nM) as
indicated. DNA from each sample was extracted, and Egfl7 promoter methylation levels
were analyzed using MSP. M: methylated; U: unmethylated. (C) miR-126 expression was
determined using qPCR and normalized to U6 expression. ADAM9 and DNMT1
expression was detected and normalized to GAPDH. The mean ± SD was determined
from 3 replicates (**P < 0.01). (D) DNMT1 expression in ECa-109 and KYSE-510 cells
transfected with miR-126 and miR-NC mimics was determined by western blotting.
Figure 4. Overexpression of miR-126 suppresses ESCC cell proliferation and
migration. (A) ECa-109 and KYSE-510 cells were transfected with miR-126 (100 nM) or
miR-NC (100 nM) mimics for 24 h, and cell viability and proliferation were assessed with
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the CCK-8 assay at 24, 48, 72, and 96 h. (B) Cell colonies were counted after transfection
for 12 days. The colony formation ratio was calculated as: (visible colony number/original
number of cells) × 100. (C) The total number of migrated cells was calculated at 48 h after
transfection. (D) ECa-109 or KYSE-510 cells transfected with miR-126 or miR-NC mimics
were injected subcutaneously into either side of the posterior flank of the same nude
mouse, respectively (n = 4). Photographs show mice and tumors 4 weeks after tumor cell
implantation. Tumor sizes are shown as the mean ± SD. Values represent the mean ± SD
(*P < 0.05; **P < 0.01; ***P < 0.001).
Figure 5. ADAM9 is the target of miR-126 in ESCC. (A) ADAM9 RNA levels in 30
matched esophageal cancer tissues and their adjacent normal tissues were determined
using qPCR and normalized to GAPDH expression. Data shown are the mean ± SD
values (***P < 0.001). ADAM9 RNA levels in cancer tissues negatively correlated with
endogenous miR-126 levels (P = 0.0019, R = -0.5446, n = 30). (B) ADAM9 protein levels
were measured in 6 matched ESCC samples and 3 cell lines by western blotting. IHC
straining for ADAM9 was performed in ECs and matched NMs. Magnification: 200×; 400×.
(C) Alignment of miR-126 with the ADAM9 3′-UTR. MiR-126 (100 nM) or miR-NC (100 nM)
mimics were co-transfected with the ADAM9-3′-UTR reporter plasmid, along with a control
Renilla luciferase pRL-TK vector, into ECa-109 or KYSE-510 cells. The relative luciferase
activity in each group was analyzed 48 h later and expressed relative to the activity in the
miR-NC-transfected group. Data are representative of 3 independent experiments. Values
represent the mean ± SD (***P < 0.001). (D) ADAM9 expression in ECa-109 and
KYSE-510 cells transfected with miR-126 mimics or siRNA against DNMT1 (si-D) was
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27
determined in by western blotting.
Figure 6. The ADAM9/EGFR/AKT signaling directly mediates the suppressive role of
miR-126 in ESCC. (A) ECa-109 and KYSE-510 cells were transfected with control siRNA
(si-Ctrl, 100 nM) or ADAM9 siRNA (si-ADAM9, 100 nM) as indicated, and cell viability was
assessed at different time points. Cell migration ability was determined with the transwell
assay, and the migrated cell number was calculated. (B) ADAM9, pEGFR, EGFR, pAKT,
AKT, pERK, and ERK protein levels were determined with western blotting and compared
to GAPDH levels. (C) ECa-109 cells were treated with an EGFR inhibitor (AG1478, 10
μM), and pEGFR, EGFR, pAKT, and AKT protein levels in ECa-109 cells were analyzed
with western blotting. Cell viability and cell migration were determined at different time
points after treatment with AG1478 (10 μM). The data are representative of 3 independent
experiments. (D) Diagram summarizing the findings. Values represent the mean ± SD (*P
< 0.05; **P < 0.01; ***P < 0.001).
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Published OnlineFirst December 15, 2014.Clin Cancer Res Ronghua Liu, Jie Gu, Pei Jiang, et al. ADAM9-EGFR-AKT signalingesophageal squamous cell carcinoma growth via DNMT1-microRNA126 epigenetic circuit contributes to
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