DNMT1-microRNA126 epigenetic circuit contributes to esophageal … · 2014. 12. 13. · Another 30...

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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].

Research. on June 12, 2021. © 2014 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 15, 2014; DOI: 10.1158/1078-0432.CCR-14-1740

http://clincancerres.aacrjournals.org/

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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




25

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




26

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




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).




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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