Background
Esophageal cancer is one of the most common cancers with high aggressiveness and poor prognosis [
1]. Esophageal squamous cell carcinoma (ESCC) is the major subtype of esophageal cancer and is highly prevalent in China [
2]. Despite substantial improvements in clinical diagnosis and therapeutics over the past two decades, the 5-year survival rate in patients with ESCC is less than 25% [
3]. There is an urgent need for understanding the molecular mechanism underlying the development and progression of ESCC.
In recent years, cancers have been widely regarded to be associated with epigenetic modifications that is defined as a stable change in gene expression without modifications in DNA sequence [
4‐
6]. DNA methylation is one of the best known epigenetic mechanisms in cancer epigenetics. DNA methylation involves in the addition of a methyl (CH3) group to DNA with DNA methyltransferases (DNMTs), thereby often modifying gene function through regulation of gene expression [
7]. DNA hypermethylation in the promotor region of tumor suppressor genes has been widely shown in various cancers [
8,
9]. Similarly, a recent study on ESCC also showed aberrant DNA methylation in the different genes including HOXB2, NEFL, SLC15A3, OBSL1 and miR-124 [
10]. However, it is lack of evidence that these genes are essentially associated with ESCC progression. Among of these genes, miR-124 caught our attention in view of the finding that large number of microRNAs(MiRNAs) have been shown important roles in the development of different cancers [
11]. In the previous studies, miR-124 has been shown to be associated with several cancers including ESCC [
10,
12]. For instance, miR-124 might have effects on decreasing risks of ESCC in subgroups of Chinese elderly persons [
13].
A microRNA is a small non-coding RNA molecule, which functions in the post-transcriptional regulation of gene expression by partially complementing with the 3′-untranslated regions (3’UTR) of specific mRNAs [
14]. According to the prediction of bioinformatics software of Miranda, we showed that 5′-UTR regions of hsa-miR-124-3p, a mature sequence of human miR-124 precursor, could bind to 3′-UTR region of branched chain amino acid transaminase 1(BCAT1) gene, the enzyme that catalyzes branched-chain alpha-keto acids to branched-chain L-amino acids essential for cell growth [
15,
16]. BCAT1 overexpression has been shown to be associated with several cancers including glioblastoma [
17], nasopharyngeal carcinoma [
18], ovarian cancer [
19] and hepatocellular carcinoma [
20].
These findings prompted us to investigate the role of DNMT/miR-124/BCAT1 axis in regulating development and progression of ESCC. With the study of loss-of-function and gain-of-function in DNMT/miR-124/BCAT1, we showed that downregulation of hsa-miR-124-3p in ESCC tissues and cells contributed to the upregulation of BCAT1 expression, which enhanced the proliferation and invasion of ESCC. Furthermore, the data demonstrated that hsa-miR-124-3p downregulation was associated with hypermethylation in its promotor region, which resulted from increased expression of DNMT1. Our study thus clarifies a new mechanism that DNMT1/miR-124/BCAT1 axis regulates the development and progression of ESCC. These findings suggest that inhibitors against the activity of DNMT1 and/or BCAT1 might be a novel strategy to inhibit development and progression of ESCC.
Methods
Patients and tissue samples
The primary ESCC tissues and adjacent normal tissues were collected from patients who underwent surgery the First Affiliated Hospital, Sun Yat-sen University. All patients have not received any therapy before surgery. The specimens were collected immediately after removing the resected esophagus, and stored at − 80 °C. The study was obtained the informed consent from all the participating patients and the approval from the Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University.
Cell culture, cell transfection and drug treatment
Human normal esophageal cell line HEEC (Het-1A, ATCC® CRL-2692™) was obtained from the America Type Culture Collection in May 2017, and two human esophageal carcinoma cell lines, KYSE-150 (TCHu236) and Eca-109 (TCHu69), were obtained from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences in May 2017. These cell lines have been authenticated by short-tandem repeat analyses. They are free of mycoplasma contamination.
The cells were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% Penicillin-Streptomycin (10,000 U/mL) (Invitrogen). For cell transfection, siRNAs including DNMT1 siRNA, BCAT1 siRNA, scrambled siRNA (NC siRNA), miR-124-3p mimics and its negative control (NC mimics) were supplied by Ribobio Company (Guangzhou, China) and pcDNA-BCAT1(OE-BCAT1) or pcDNA-negative (OE-NC) were constructed using pcDNA3.1(+) vector. KYSE-150 and Eca-109 cells grown in 6-well cell plates (1 × 106) were transfected with siRNA or pcDNA3.1vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. After 6 h, the transfection solution was replaced by complete medium.
For drug treatment, KYSE-150 or Eca109 cells were treated with 5 μM 5-Azacitidine (5-Aza, Sigma-Aldrich, Saint Louis, MO, USA), which is the inhibitor of DNMT1.
Real-time PCR
Total RNA from tissue samples and cells were extracted with RNAprep pure tissue kit (Tiangen Biotech Co., LTD., Beijing, China) and Rneasy Mini Kit (Qiagen, Germantown, MD, USA) respectively. The mRNA of DNMT1, miR-124-3p and BCAT1 were amplified by quantitative real-time RT-PCR. It was carried out using SuperScriptIII Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen) by an ABI 7500 Fast real-time PCR instrument (Applied Biosystems, Foster City, CA, USA). It was carried out with the following procedures: 50 °C for 3 min, 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 30 s. The specific primers used in quantitative real-time RT-PCR were displayed as follows: DNMT1: forward, 5′-CCTAGCCCCAGGATTACAAGG-3′; reverse, 5′-ACTCATCCGATTTGGCTCTTTC-3′; BCAT1: forward: 5′- GAGCCTGGAAAGGTGGAACTG-3′; reverse, 5′- GCTGACACCCATTATCTACTGCT-3′; GAPDH: forward, 5′-TGTTCGTCATGGGTGTGAAC-3′; reverse, 5′-ATGGCATGGACTGTGGTCAT-3′; hsa-miR-124-3p: forward, 5′-ACACTC CAGCTGGGTAAGGCACGCGGTGAA-3′; reverse, 5′- CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGCATTCAC-3; U6: forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The data were analyzed according to the comparative Ct method.
Bisulfite sanger sequencing
Genomic DNA from ESCC tissues or adjacent normal tissues was extracted using QIAamp DNA Mini Kit (Qiagen) and bisulfite converted using an EpiTect Bisulfite Kit (QIAGEN). The hsa-miR-124-3p was enriched by PCR with Taq DNA Polymerase (Invitrogen) and primer designed using Pyrosequencing Assay Design Software (Qiagen). The PCR products were cloned into pGEMT vector, then subjected to Sanger sequencing. Five single molecules were sequenced for each sample.
Methylation specific PCR (MSP)
The bisulfite converted DNAs were used as the templates for the MSP assay, with miR-124-3p primers that distinguish the methylated (M) from unmethylated (U) DNA: M forward: 5′-GTATTTGGGGGTTTATTTTTTGTC-3′; M reverse: 5′- GAAACCGACTCGAACTTACGTA-3′; U forward: 5′-GTATTTGGGGGTTTATTTTTTGTT-3′; U reverse: 5′-ATCAAAACCAACTCAAACTTACATA-3′. PCR amplification was conducted at 95 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 56 °C for 1 min, 72 °C for 30 s, and the elongation at 72 °C for 5 min. A plasmid containing methylated miR-124-3p sequence and water without DNA template were used as positive control and negative control, respectively. Finally, MSP products were analyzed using agarose gel electrophoresis.
Prediction of hsa-miR-124-3p targets
The target of hsa-miR-124-3p was predicted with the online software Miranda, which is an algorithm for determining potential target sites of microRNA in genomic sequences. Briefly, hsa-miR-124-3p sequences were first scanned in all genomic DNA/RNA sequences. Then, the potential target sites were identified with a dynamic programming local alignment and the thermodynamic stability estimation of RNA duplexes. The detected target with energies less than an energy threshold was identified as potential target sites.
Luciferase assay
The human BCAT1 3′-UTR DNA sequence that was predicted to interact with miR-124-3p and the mutant BCAT1 3′-UTR DNA sequence were amplified and inserted into psi-CHECK2 renilla/firefly dual-luciferase expression vector (Promega, Mullion, WI, USA), with the following primers: BCAT1-WT-1: 5′-CCGCTCGAGTTTGAAGGTCCTCCAAGTCCGTGT-3′ (forward) and 5′-ATTTGCGGCCGCCTAATGCTGCCACTGAGCTGACAAG-3′ (reverse). BCAT1-MUT-1: 5′-AAGACTGAGGGTCTTCGCATACATAGATCTTTGTATC-3′ (forward) and 5′-GATACAAAGATCTATGTATGCGAAGACCCTCAGTCTT-3′ (reverse). BCAT1-WT-2: 5′-CCGCTCGAGGAGGGTGATGACATACATTTACTGG-3′ (forward) and 5′-ATTTGCGGCCGCAACTTTTCAGAATCCCTAGTGGC-3′ (reverse). BCAT1-MUT-2: 5′-ATTCCATATACATATCGCATACATATTGTGATAATTT-3′ (forward) and 5′-AAATTATCACAATATGTATGCGATATGTATATGGAAT-3′ (reverse). KYSE-150 or Eca-109 cells were co-transfected with the BCAT1-WT/BCAT1-MUT reporter constructs, and miR-124-3p mimics/NC mimics with Lipofectamine 2000 (Invitrogen). Luciferase activities were determined after 48 h using the Dual-Luciferase reporter assay system (Promega) on Enspire. Data are presented as the ratio of renilla luciferase activity to firefly luciferase.
Immunohistochemistry staining
ESCC tissue or adjacent normal tissue samples were fixed in 10% neutral buffered formalin (NBF). Thereafter, the tissues were paraffin-processed and embedded. The deparaffinized tissue sections (4 mm thick) were stained with antibody against BCAT1 (Santa Cruz, Dallas, Texas, USA) for immunohistochemical analysis. Images of immunohistochemistry staining were photographed under a light microscope (Leica, Wetzlar, Germany).
Western blot analysis
Total cell proteins were extracted using the mammalian protein extraction reagent including halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentrations were measured by BCA protein assay kit (Thermo Fisher Scientific) and equal amount of proteins were subjected to SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against DNMT1, BCAT1, and GAPDH (Santa Cruz), respectively, and followed by incubation with the HRP-conjugated secondary antibodies. The signals were detected using an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
Immunofluorescence staining
KYSE-150 or Eca-109 cells grown on coverslips were treated with miR-124-3p mimics/NC mimics or BCAT1 siRNA/NC siRNA for 48 h. Then the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. After blocked with 3% bovine serum albumin, cells were incubated with primary antibodies against DNMT1, BCAT1 and DAPI. Subsequently, cells were stained with the Alexa Fluor 488-conjugated and FITC-conjugated secondary antibodies (BD Biosciences, San Diego, CA, USA). Finally, immunofluorescence images were visualized under LSM 510 Meta confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany).
CCK-8 assay
Cell viability was measured using Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). KYSE-150 or Eca-109 cells grown in 96-well plates were treated with miR-124-3p mimics/NC mimics or BCAT1 siRNA/NC siRNA for 24 h, 48 h or 72 h. After washing with PBS for three times. A 1:10 diluted CCK-8 solution was added and incubated at 37 °C for 2 h. The absorbance was determined at 450 nm on Enspire (PerkinElmer, Waltham, MA, USA).
For colony formation assay, KYSE-150 or Eca-109 cells treated with miR-124-3p mimics/NC mimics or BCAT1 siRNA/NC siRNA were seeded in a 3 mm2 plate. After 2 weeks of incubation, plates were washed with PBS gently and stained with 0.5% crystal violet in 20% ethanol for 15 min at room temperature. Lastly, the cells were imaged after rinsed with PBS.
Transwell assay
The invasion capability of cells was determined by the transwell assay. After transfection for 48 h, KYSE-150 or Eca-109 cells in serum-free media were transferred into the upper chamber of Transwell assay inserts (Millipore) and media supplemented 10% FBS was added to the bottom chamber. After incubation at 37 °C for 24 h, the invading cells through the membrane were stained with 0.5% crystal violet in 20% ethanol and photographed under the light microscope.
Discussion
MiR-124 has been shown to function as a tumor suppressor in esophageal cancer [
12]. However, it is not clear concerning the mechanism by which miR-124 expression was downregulated in esophageal cancer and its subtype ESCC. In addition, there is still lack of direct experiment evidence suggesting that miR-124 as a tumor suppressor regulates the initiation and development of ESCC. We herein showed that significantly increased expression of hsa-miR-124-3p was observed in the paracancerous normal tissues of ESCC and the normal cell line HEEC compared to ESCC tissues and two ESCC cell lines. In addition, over-expression of hsa-miR-124-3p is highly negatively correlated with proliferation and migration in several ESCC cells in the present study. These findings imply that decreased expression of hsa-miR-124-3p might be highly correlated with initiation and progression of ESCC.
The first crucial insight gleaned from our present study is that although miR-124 has been reported to target several associated-cancer genes including RAC1, ROCK1 [
23], STAT3, the androgen receptor [
24], PIK3CA [
25], EZH2 [
26] and CBL [
27], we showed a new target BCAT1 gene of hsa-miR-124-3p through bioinformatics prediction and dual-Luciferase reporter assay. The BCAT1 gene, known previously as ECA39, is an enzyme transforming α-amino groups of branched chain amino acids (BCAAs) to a-ketoglutarates (a-KG) [
15,
16], yielding glutamate frequently required for the growth of malignant cells [
28]. BCAT1 has been shown overexpression in some human malignancies, which is highly correlated with initiation and progression of malignant tumor [
17,
18,
29]. However, to the best of our knowledge, there is no report showing association of BCAT1 overexpression with ESCC. We thus speculated that growth and migration inhibition in ESCC cells mediated by hsa-miR-124-3p was associated with downregulation of BCAT1 expression. Consistent with this, our results indicated that BCAT1 knockdown significantly inhibited cell proliferation and migration of ESCC cell lines KYSE-150 and Eca109.
Regulation of BCAT1 expression in different tumors involves in different mechanisms. The aberrant DNA methylation in
BCAT1 promotor region was associated with in colorectal cancer, ovarian cancer and gliomas [
17,
30]. These findings suggest that epigenetic mechanisms could account for altered BCAT1 expression in different cancer types, including EOC. In the present study, however, we showed that BCAT1 expression was directly regulated by hsa-miR-124-3p since hsa-miR-124-3p bound to 3′-UTR region of BCAT1 gene, degrading BCAT1 mRNA. In fact, we also observed downregulation of BCAT1 expression in KYSE-150 and Eca109 cells treated with hsa-miR-124-3p mimics. It should be also noted that BCAT1 expression might be also regulated by DNA methylation in BCAT1 promotor region in KYSE-150 and Eca109 cells although we did not provide the direct evidence. This is because expression of DNMT1, an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA, significantly increased in ESCC tissues and two ESCC cell lines.
Some miRNAs containing CpG islands are susceptible to methylation-associated silencing [
31‐
33]. Methylation-associated silencing of tumor-suppressive miRNAs might play a crucial role in the tumorigenesis through activating oncogenic pathways. MiR-124, as a typical tumor-suppressive miRNA, has also been found epigenetically silenced in cholangiocarcinoma, cervical cancer and pancreatic cancer [
34‐
36]. Similarly, a recent study showed that miR-124 gene were highly methylated in LNM-positive ESCC [
10]. Those findings are in agreement with our results showing hypermethylation miR-124 gene in ESCC tissues and two ESCC cell lines. However, in the previous study it is unclear that hypermethylation in miR-124 gene was linked to overexpression of DNMT in ESCC. In the present study, we provide the direct evidence suggesting that hypermethylation in miR-124 gene was strongly mediated by DNMT1 through DNMT1 knockdown and 5-AZ treatment. This finding is consistent with the previous study showing DNMT1-mediated downregulation of miR-124 expression in the intrahepatic cholangiocarcinoma [
34]. Furthermore, our results demonstrated that DNMT1 knockdown or 5-AZ treatment significantly inhibited cell proliferation and migration of ESCC cell lines KYSE-150 and Eca109 through increasing hsa-miR-124-3p expression and inhibiting downstream BCAT1 expression.
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