Epigenetic silencing of miRNA‑9 is correlated with promoter‑proximal CpG island hypermethylation in gastric cancer in vitro and in vivo

  • Authors:
    • Yan Li
    • Zhong Xu
    • Bo Li
    • Zhengzheng Zhang
    • Hongchun Luo
    • Yuanhu Wang
    • Zhizhong Lu
    • Xiaoling Wu
  • View Affiliations

  • Published online on: September 23, 2014     https://doi.org/10.3892/ijo.2014.2667
  • Pages: 2576-2586
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Silencing of protein‑coding tumor suppressor genes (TSGs) by CpG island hypermethylation is a common occurrence in gastric cancer (GC). Here, we examine if tumor suppressor microRNAs (miRNAs) are silenced in a similar manner. Real‑time quantitative PCR (RTQ‑PCR) was employed to investigate the expression level of four candidate miRNAs in GC tissues (n=30) and cell lines. Basing on RTQ‑PCR results and bioinformatics approach, miR‑9 was chosen for further study on epigenetic regulation. Bisulfite genomic sequencing PCR (BSP) was performed to assess the methylation status of miR‑9 in GC tissues. In both GC cell lines and animal models, demethylation was performed either by treatment with 5‑aza‑2'‑deoxycytidine (5‑AZA‑CdR) or by siRNA targeting DNMT1. We also analyzed the relationship between miRNAs and several clinicopathological features. Candidate miRNAs (miR‑9, miR‑433, miR‑19b, and miR‑370) were found strongly downregulated in GC tissues and cell lines. Their expression was increased following 5‑AZA‑CdR treatment. CpG island methylation of miR‑9 was significantly higher in GC tissues compared to normal controls. After two demethylation treatments, miR‑9 methylation degree was significantly decreased and miR‑9 expression was ob­viously restored in GC cells and animal models. Deregulation of miR‑9 was positively correlated with tumor lesion size. Three other miRNAs, miR‑19b, miR‑433, and miR‑370 were assοciated with lymph node metastasis, decreased curvature, and poorly differentiated carcinoma. miR‑19b and miR‑433 were positively correlated with male gender. Of four candidate miRNAs downregulated in GC, miR‑9 is epigenetically regulated by DNA methylation both in vitro and in vivo.

Introduction

microRNAs (miRNAs) are a class of small, endogenous, non-coding RNA molecules that are typically 20–25 nucleotides in length. miRNAs negatively regulate specific gene products by translational repression or mRNA degradation via binding to partially or perfectly complementary sequences in the 3′ untranslated regions of target genes (13).

In human tumors, some miRNAs are upregulated and function as oncogenes, while others are downregulated functioning as tumor suppressor genes (TSGs). Recent studies have shown that 50% of miRNAs are located within fragile sites, thus supporting the fact that many of these miRNAs may be lost during tumorigenesis (1,414). Consistent with this, significant data indicate that many miRNAs exhibit decreased expression in tumors (1,5,6,8,10,13,1525). Thus, although miRNAs have been shown to be both pro- and anti-tumorigenic, the majority seems to function as TSGs by negatively regulating protein-coding oncogenes and genes regulating cell proliferation and apoptosis (1,13,16,2022,2630).

The promoter regions of many genes, including a number of TSGs, sometimes are embedded in CpG islands regions within the DNA that are subject to methylation. In normal condition, these regions tend to be unmethylated. However, in a transformed setting, many of these CpG islands become hypermethylated, resulting in silencing of gene expression. Although hypermethylations of CpG islands has been mostly described for protein-coding genes, a similar mechanism may be responsible for silencing expression of miRNAs that possess antitumorigenic properties; a mechanism such as this could potentially enhance tumorigenesis (13,16,21,22,3037).

Gastric cancer (GC) has a poor prognosis, in large part, because patients often present with advanced disease. Limitations of early diagnosis and effective therapies unfortunately result in high lethality. Thus, additional research to improve both detection and treatment of GC is critical. Several studies have been performed examining the miRNA expression profile of multiple tumor types (6,11,14,20,27, 28,34,3842). Evidence suggests that hypermethylation of CpG islands related with the promoters of miRNA genes is a common event in GC (20,28,34,37,38,40,4248). Previously, we examined several miRNAs, including 19 downregulated and seven upregulated, in GC (6). Here, we specifically follow up on the downregulated miRNAs and investigate the mechanism of their decreased expression. Four down-regulated miRNAs contain CpG islands within 5,000 bp upstream of the transcriptional start site, and these were selected as initial candidate genes. We measured miRNA expression levels in GC samples to validate the miRNA expression profile data. To assess the importance of methylation in expression of these genes, we treated GC cells with a demethylating agent. Based on these initial results, miR-9 was selected for additional epigenetic research. Thus, we studied the role of promoter methylation in regulating miR-9 expression both in vitro and in vivo.

Materials and methods

Cell lines and animals

Cell lines used in this study included human GC cell lines SGC-7901 and BGC-823 and normal gastric epithelium cell line GES-1. The cells were purchased from the Centre of Cell Cultures of Chinese Academy of Medical Sciences, Shanghai, China. All cell lines were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM gluta-mine, and 1 mM sodium pyruvate. Cells were housed in humidified incubators at 37°C in an atmosphere with 5% CO2. Cells were maintained as a monolayer by serial passaging after trypsinization with 0.1% trypsin (Beyotime, Jiangsu, China).

Five to six-week-old male Balb/c nu-nu mice, weighing 18–20 g, were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Medical Sciences, China. They were maintained in cages in a pathogen-free environment (temperature 25–27°C, humidity 45–50%) and supplied with food and water ad libitum. All animals received humane care in accordance with institutional policies on Human Care and Use of Laboratory Animals and with the approval of the Ethics Committee of Chongqing Medical University.

GC samples

A total of 30 GC samples were obtained via surgery from patients that had provided informed consent to the General Surgery Department of the Second Affiliated Hospital, Chongqing Medical University, Chongqing, China. Matched controls (non-cancer gastric mucosa) were obtained from all patients. None of the patients had received any pre-operative treatment. Clinicopathologic information, such as age, gender, stage, grade, pathological diagnosis, and lymph node metastasis, was available. The study was approved by the Ethics Committee of Chongqing Medical University.

miRNA microarray and bioinformatics

We previously profiled GC samples for miRNA expression by microarray analysis (6). Data from that experiment prompted us to focus on miRNAs that were decreased in GC since these miRNAs may be functioning as tumor suppressors in the disease.

CpG Island Searcher (http://cpgislands.usc.edu/) and CpG plot (http://www.ebi.ac.uk/emboss/cpgplot) were used to determine which miRNAs were embedded in or located near (<500 bp 5′-upstream) a CpG island. Over 90% of human miRNA promoters are located 1,000 bp upstream of the mature miRNA (16,31). Promoter miRNA gene clusters were predicted using a combination of Promoter 2.0 (http://www.cbs.dtu.dk/services/Promoter/), Promoter Scan (http://www-bimas.cit.nih.gov/molbio/proscan/), and Neural Network Promoter Prediction (NNPP) (http://www.fruitfly.org/seq_tools/promoter.html).

RNA isolation and reverse transcription

Total RNA was extracted using TRIzol (Sigma-Aldrich Chemical Co., Milwaukee, WI, USA). Concentration and purity were assessed with an ultraviolet spectrophotometer at wavelengths of 260 and 280 nm. RNA was reverse transcribed into cDNA using the reverse transcription kit (Takara Bio, Inc., Dalian, China). RT primers are listed in Table I. A master mix (20 μl total) containing 5× PrimeScript Buffer (4 μl), PrimeScript RT Enzyme Mix I (1 μl), RT specific primer (1 μl), total RNA (1 μg), and nuclease-free water (13 μl) was prepared on ice. The reaction was performed at 42°C for 15 min followed by 85°C for 5 sec.

Table I

Reverse transcription primers.

Table I

Reverse transcription primers.

Gene namePrimer sequence
U6 5′-CGCTTCACGAATTTGCGTGTCAT-3′
hsa-miR-9 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTCATACAG-3′
hsa-miR-433 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACACCG-3′
hsa-miR-19b 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTCAGTT-3′
hsa-miR-370 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACCAGG-3′
Real-time quantitative PCR (RTQ-PCR)

RNA samples isolated from both GC tissues (n=30) and cell lines were converted into cDNA and analyzed by RTQ-PCR. GC cell lines and tumors from animal models following treatment with 5-aza-2′-deoxycytidine (5-AZA-CdR) or transfection with siRNA-DNMT1 were also examined by this method. RTQ-PCR was performed using the SYBR-Green real-time PCR master mix kit (Takara Bio, Inc.) and the IQ5 PCR instrument. In brief, a master mix (25 μl) was prepared on ice with 12.5 μl 1× SYBR-Green buffer, 1 μl each primer, 2 μl cDNA, and 8.5 μl nuclease-free water. The cDNA was initially denatured at 95°C for 30 sec followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 59°C for 30 sec, and extension at 72°C for 30 sec. Primer sequences were designed using software Primer 5.0, and sequences are listed in Table II. U6 snRNA served as an endogenous control. All experiments were performed in triplicate.

Table II

RTQ-PCR primers.

Table II

RTQ-PCR primers.

Gene namePrimer sequenceAnnealing temperature (°C)Product length (bp)
U6F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′
R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′
5989
hsa-miR-9F: 5′-GGGTCTTTGGTTATCTAGC-3′
R: 5′-TGCGTGTCGTGGAGTC-3′
5963
hsa-miR-433F: 5′-GGATCATGATGGGCTGGT-3′
R: 5′-CAGTGCGTGTCGTGGAGT-3′
5964
hsa-miR-19bF: 5′-CGTGTGCAAATCCATGC-3′
R: 5′-CAGTGCGTGTCGTGGAG-3′
5965
hsa-miR-370F: 5′-GCCTGCTGAGATGGAATCTGATGTC-3′
R: 5′-CCAGTGCGTGTCGTAGAGTCATCAA-3′
5963

[i] RTQ-PCR, real-time quantitative PCR.

Each run was accompanied by a melting curve analysis to confirm the specificity of amplification and absence of primer dimers. Relative quantification of miRNA expression was calculated by the 2−ΔΔct method.

Bisulfite genomic sequencing PCR (BSP)

DNA was isolated from GC tissues (human samples, n=30; animal samples, n=10) and cell lines (including matched normal controls). GC cell lines and tumor tissues from animal models (n=10) following 5-AZA-CdR treatment or siRNA-DNMT1 transfection were also included. Genomic DNA was isolated using the Genomic DNA Extraction kit (Takara Bio, Inc.) according to the manufacturer’s instructions. Bisulfite modification (EZ DNA Methylation-Gold™ kit, D5005/50; Zymo Research Corp., Irvine, CA, USA) was performed to convert unmethylated cytosine to uracil; methylated cytosine nucleotides are unaffected by the procedure. Bisulfite-modified miRNA promoters were amplified using specially designed primers listed in Table III. The reaction volume 50 μl included 10× buffer (5 μl), MgCl2 (2 μl), dNTP (1 μl), each primer (2 μl), DNA (5 μl), Platinum Taq (0.3 μl), and ddH2O (32.7 μl). Amplification was carried out as follows: 5 min 95°C; 42 cycles of 30 sec at 95°C, 30 sec at 57°C, and 40 sec at 72°C; and a 10 min final extension at 72°C. Per sample, five independent colonies for each tested region were picked and sequenced. The extent of methylation was assessed by identifying the number and position of methylated cytosine residues.

Table III

Specific BSP primers for miRNA-9.

Table III

Specific BSP primers for miRNA-9.

Gene namePrimer sequenceProduct length (bp)Tm (°C)
miR-9-1F: 5′-GGTAGAGTTAATTAGAGGATGGTTTG-3′
R: 5′-ACCAAAAATCACCCAAAATTATAAA-3′
49857
miR-9-2F: 5′-TGATTTTTGGTTTTTTTTGAAT-3′
R: 5′-TCCACTACCCTTCTCTAAAAAA-3′
50458

[i] BSP, bisulfite genomic sequencing PCR.

5-AZA-CdR treatment in vitro and in vivo

Cell lines were treated with 5 μM 5-AZA-CdR (Sigma-Aldrich Chemical Co.) for 48 h.

Nude mice were subcutaneously injected with 107 GC cells on either side of the flank. Two weeks post-implantation, tumors reached a size of ~1.2 cm3. Orthotopic GC models (n=10) were constructed according to our previously described protocol (49). Animals received intravenous injection of 5-AZA-CdR at 0.6 mg/kg once per day for 4 weeks after transplantation (50). Mice were euthanized and fresh tumor fragments, free of any necrotic region, were harvested and preserved in liquid nitrogen until use.

siRNA-DNMT1 synthesis and transfection into cell lines and animal models in vivo

An siRNA-DNMT1 plasmid was synthesized by Jingsai Bio Co., Ltd., (Hubei, China). Gastric cell lines were seeded at a density of 2×105 cells/well in 6-well plates (Costar, Cambridge, MA, USA) and cultured overnight. siRNA-DNMT1 plasmid (5 μl) was transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were incubated for 48 h, at which time, the transfection efficiency was >80%.

Animal models (n=10) using transfected GC cell lines were established as described above. Four weeks after implantation, fresh tumor tissues were harvested and preserved as described.

Statistical analysis

Data are presented as mean ± SD. RTQ-PCR data were analyzed by Student’s t-test. The correlation between miRNA expression and clinicopathological factors was analyzed using Fisher’s exact test. Differences in mean methylation levels were analyzed using the χ2 test. Statistical significance was set at p<0.05.

Results

Selection of candidate miRNAs of interest

We previously profiled GC samples for miRNA expression by microarray analysis (6). A total of 26 differentially expressed miRNAs were identified, of which 19 were downregulated and seven were upregulated. We prioritized studying miRNAs that were repressed in GC and that had CpG islands within 5,000 bp upstream of the transcription start site. The final list of potential target genes was determined using a bioinformatics approach (51). Potential targets included miR-9, miR-433, miR-19b, and miR-370 (Fig. 1A–D).

Validation of expression of four miRNAs in GC tissues and cell lines

We measured expression of miR-9, miR-433, miR-19b, and miR-370 and found that they were all strongly repressed in GC samples compared to normal gastric mucosa. All miRNAs, except miR-19b, displayed significant differences in expression (p<0.05) (Fig. 2A). Compared to the normal gastric epithelial cell line GES-1, the four tested miRNAs all showed decreased expression in the GC cell lines SGC-7901 and BGC-823. However, miR-9 was the only miRNA of the four whose expression level was statistically significantly decreased (p<0.05) (Fig. 2B).

miRNA expression is increased following treatment with demethylating agent

To assess the importance of methylation in expression of the four candidate miRNAs, we examined their expression in two GC cell lines following treatment with 5-AZA-CdR. In both cell lines, miRNA expression was increased after demethylation. In SGC-7901, miR-9 and miR-19b were both significantly increased following 5-AZA-CdR treatment (p<0.01); in contrast, the increased expression of miR-370 and miR-433 was not statistically significant (Fig. 3A). In the BGC-823 cell line, statistical significance was achieved for all four miRNAs (p<0.01) (Fig. 3B).

Analysis of DNA methylation in the miR-9 CpG island

Approximately 90% of human miRNA promoters are located 1,000 bp upstream of the mature miRNA (16,31). To identify promoters harboring CpG islands, a manual search of the candidate miRNA promoters was performed via a bioinformatics approach. miR-9 promoter was predicted to embed in CpG islands based on this analysis (Fig. 4). Moreover, we found that miR-9 was consistently deregulated in both GC tissue and cell lines. Thus, miR-9 was chosen for further study.

We investigated the methylation status of the miR-9 promoter (500–1,500 bp upstream of the transcription start site) via amplification of two regions termed miR-9-1 and miR-9-2. CpG island methylation of miR-9 in GC tissues was significantly higher than the methylation level in normal gastric mucosa (p<0.05 and p<0.005) (Fig. 5A and B). Cell line data were consistent with this. That is, miR-9 methylation in the GC cell lines SGC-7901 and BGC-823 was significantly higher than methylation in normal controls (p<0.01 and p<0.005) (Fig. 5C and D).

Demethylation, induced by either 5-AZA-CdR or siRNA-DNMT1, increases miR-9 expression in GC cell lines

We assessed the importance of miR-9 methylation on its expression by two methods. First, SGC-7901 and BGC-823 cells were treated with 5-AZA-CdR. In both cell lines, methylation of miR-9 CpG islands was decreased following treatment (p<0.05, p<0.005) (Fig. 6A and B). This was concomitant with increased miR-9 expression (p<0.01) (Fig. 4A and B). SGC-7901 and BGC-823 cells were also transfected with siRNA targeting DNMT1 as another means of relieving DNA methylation. Consistent with the 5-AZA-CdR results, transfection of siRNA-DNMT1 decreased CpG island methylation in both cell lines (p<0.05) (Fig. 6C and D) and increased expression of miR-9 (p<0.01) (Fig. 6E and F).

miR-9 methylation in the GC animal model

We next explored miR-9 methylation in our orthotopic GC animal model. In tumors, the degree of methylation of the miR-9-1 CpG island was decreased following 5-AZA-CdR treatment, but the change was not statistically significant. In contrast, methylation of miR-9-2 was significantly decreased by 5-AZA-CdR treatment (p<0.01) (Fig. 7A). Although expression of miR-9 showed an increasing trend following treatment, the change was not significant (Fig. 7B). Introduction of siRNA-DNMT1 into our GC animal models significantly decreased the levels of methylation of both miR-9-1 (p<0.01) and miR-9-2 (p<0.05) (Fig. 7C). This occurred concomitantly with increased miR-9 expression (p<0.01) (Fig. 7D).

Correlation between miRNAs and clinicopathological features

We performed correlation analysis between miRNA expression and several clinicopathological features, and the data are listed in Table IV. Of note, deregulation of miR-9 was positively correlated with tumor size (p=0.026) and lymph node metastasis (p=0.041). miR-433 correlated with gender (p=0.031), the position of local invasion (p=0.006), grade (p=0.006), and lymph node metastasis (p=0.003). miR-19b was found to correlate with gender (p=0.031), the position of tumor involvement (p=0.001), and grade (p=0.031). Finally, miR-370 correlated with tumor position (p=0.001), grade (p=0.031), and lymph node invasion (p=0.012).

Table IV

Correlation between miRNA and clinicopathological features in gastric cancer (n=30).

Table IV

Correlation between miRNA and clinicopathological features in gastric cancer (n=30).

Clinicopathological featuresCasesmiR-9p-valuemiR-433p-valuemiR-19bp-valuemiR-370p-value




LEHELEHELEHELEHE
Gender
 Male232210.128221a0.031221a0.0312120.225
 Female752434352
Age
 ≤50 years101000.281910.640820.407820.407
 >50 years20173164182182
Tumor size
 ≤3 cm1394a0.0261030.3671210.4091210.409
 >3 cm17170152143143
Tumor position
 Lesser curvature232210.128221b0.006230b0.001230b0.001
 Greater curvature752343434
Pathological pattern
 Squamous carcinoma000None00None00None00None
 Adenocarcinoma30273255264264
Pathological grade
 Well differientiated7520.12834b0.00643a0.03143a0.031
 Poorly differientiated23221221221221
Lymph node metastasis
 Yes19190a0.041190b0.0031810.126190a0.012
 No1183658374
Liver metastasis
 Yes000None00None00None00None
 No30273255264264
Peritoneum dissemination
 Yes000None00None00None00None
 No30273255264264
Clinical stages
 I, II292630.9002450.8332540.8672540.867
 III, IV110101010

a P<0.05,

b p<0.01.

{ label (or @symbol) needed for fn[@id='tfn5-ijo-45-06-2576'] } miRNA, microRNA; LE, low expression; HE, high expression.

Discussion

miRNAs are heavily implicated in tumorigenesis in multiple cancer types (714). We identified four candidate miRNAs (miR-9, miR-433, miR-19b, and miR-370) whose expressions were all reduced in GC tissues and cell lines compared to normal healthy gastric epithelium. All of the miRNAs, except for miR-19b, displayed significant differences in expression, validating the previous miRNA profile data from GC patients. The only miRNA that showed consistent downregulation in GC cell lines SGC-7901 and BGC-823 (as in GC tissue) was miR-9. Recently, Du et al showed that miRNAs in GC cell lines might not be repressed to the same extent as they are in actual human tissue samples; in fact, they state that most cell lines likely exhibit normal miRNA expression (28). Their study may provide an explanation as to why miR-433, miR-19b, and miR-370 were not significantly repressed in the cell lines we examined. Many other groups have identified miRNAs that are specifically downregulated in GC samples (14,20,27,28,38,42,43,47).

The four candidate miRNAs included in this study had CpG islands within 5,000 bp upstream of the transcriptional start site. Thus, we hypothesized that expression of these miRNAs would be increased following treatment with the demethylation agent 5-AZA-CdR. This proved true, as expression of all four miRNAs (miR-9, miR-433, miR-19b, and miR-370) were increased after 5-AZA-CdR treatment; thus, methylation is an important epigenetic regulatory mechanism governing expression of these miRNAs. In both SGC-7901 and BGC-823 cells, the effect of 5-AZA-CdR on miR-9 and miR-19b was the greatest, suggesting that these two miRNAs are dominantly regulated by a methylation-dependent mechanism. Interestingly, we found that the ability of miR-370 and miR-433 to be demethylated was different in different GC cell lines. Consistent with this, Guo et al reported that, following 5-AZA-CdR treatment of other GC lines (HGC-27 and MGC-803), miR-433 was re-expressed to different degrees (38). Thus, aberrant expression of miRNAs in different tumor cell lines may result from tumor heterogeneity (28). One study showed that miRNA expression could be rescued by deacetylation even if miRNA hypermethylation was maintained (25). The exact role of DNA hypermethylation of miR-370 and miR-433 in SGC-7901 cells requires additional research.

In cancer, many protein-coding genes with tumor suppressor qualities are silenced by CpG island methylation. Here, we examined if tumor suppressor miRNAs may be silenced in a similar manner. Methylation of miR-9 at two promoter CpG islands was significantly higher in GC tissues and cell lines compared to normal controls. This finding supports our hypothesis that tumor suppressor miRNAs can be silenced by DNA methylation in tumors, similar to the silencing of protein-coding genes. We found that miR-9 is epigenetically regulated by hypermethylation of promoter-proximal CpG islands; this may be the dominant mechanism of miR-9 silencing in GC.

miR-9 is the best characterized miRNA regulated by methylation in cancer. Lehmann et al (35) showed that deregulation of hsa-miR-9-1, mediated by CpG island methylation, was an early event during breast tumorigenesis. Lujambio et al (31) determined that methylation-mediated silencing of the miR-148a, miR-34b/c, and miR-9 promoters was cancer-specific and closely correlated with lymph node metastasis. Du et al (28) found that hypermethylation repressed expression of seven miRNAs in GC; interestingly the degree of miR-9 methylation was the most significant among them. Another study also showed that miR-9 methylation correlated with decreased expression in GC (20). Taken together, a number of studies have shown the importance of miRNA methylation in GC development (20,34,38,52,53). The data we present here contribute to this base of knowledge.

We validated, by two independent methods, that miR-9 expression is epigenetically regulated in vitro. The degree of CpG island methylation of miR-9 was significantly decreased after 5-AZA-CdR treatment or siRNA-DNMT1 in both GC cell lines and animal models; this was concomitant with an increase in miR-9 expression. Our data are supported by the study of others highlighting the importance of miRNA methylation (7,8,10,5456).

We examined the importance of miR-9 methylation both in cell lines and in animal models. In all cases, miR-9 methylation was decreased and expression was increased following administration of a demethylating agent. However, the changes in miR-9 methylation and expression in vivo were not as dramatic as the in vitro alterations. The discrepancies could be due to the method of 5-AZA-CdR treatment in vitro versus in vivo, including dosage, duration, and pathway. While some have reported on 5-AZA-CdR treatment in mice, it is difficult to evaluate the effects in different laboratories (57). To the best of our knowledge, this is the first report describing the use of a demethylation drug in a GC animal model. Administration of 5-AZA-CdR in GC animal models likely requires further optimization. Although there were some differences in treatment with 5-AZA-CdR compared to siRNA-DNMT1, we effectively showed that miR-9 is epigenetically regulated by hypermethylation in GC.

We found that our four candidate miRNAs were significantly positively correlated with several clinicopathological features. Low levels of miR-9 were associated with tumor size, indicating that miR-9 may be an independent diagnostic factor in GC. Deregulated expression of miR-9, miR-433, and miR-370 was correlated with lymph node metastasis, which contributed to poor prognosis. In addition, decreased expression of these three miRNAs (miR-19b, miR-433, and miR-370) was shown to be associated with less curvature of the stomach; this shape is the most common position for both GC and poorly differentiated carcinoma, which is the most common pathological type of late stage GC. miR-19b and miR-433 positively correlated with male gender, inferring that the aberrant expression of these two miRNAs might be common events in male GC patients. Recently, Tsai et al showed that miR-9 expression was associated with tumor grade, metastasis, and survival rate in GC (20). Yanaihara et al (4) examined 104 lung cancer samples and analyzed potential correlation between six over-expressed miRNAs (miR-205, miR-99b, miR-203, miR-202, miR-102, and miR-204-prec) and clinicopathological features; however, no miRNA was found to be associated with gender. Similar analyses have been performed in multiple tumor types (4,12,13).

In conclusion, we showed that four miRNAs were downregulated in GC, and that their expression can be restored following treatment with 5-AZA-CdR. Additionally, these four miRNAs were positively correlated with several clinicopathological features. Of the four candidate miRNAs, miR-19b and miR-370 are the least studied in GC. To strengthen our claims, we performed experiments with two different demethylation methods; together, our data support that miR-9 is epigenetically silenced by CpG island methylation in GC. Due to technical limitations, the in vivo experiments should be revisited in more detail in the future. Future studies should also focus on how miRNAs regulate oncogenesis and which genes are the key miRNA targets.

Acknowledgements

We thank Professor Like Xiang for collecting the GC samples. We also thank Juan Luo (MD) for technical advice on treating GC samples and Xingxing Li (MD) and Yingying Zhang (MD) for assistance with cell biology techniques. We are grateful to Xiong Zhang (MM) for help with primer design.

References

1 

Zhang B, Pan X, Cobb GP and Anderson TA: microRNAs as oncogenes and tumor suppressors. Dev Biol. 302:1–12. 2007. View Article : Google Scholar : PubMed/NCBI

2 

Finnegan EJ and Matzke MA: The small RNA world. J Cell Sci. 116:4689–4693. 2003. View Article : Google Scholar : PubMed/NCBI

3 

Weber B, Stresemann C, Brueckner B and Lyko F: Methylation of human microRNA genes in normal and neoplastic cells. Cell Cycle. 6:1001–1005. 2007. View Article : Google Scholar : PubMed/NCBI

4 

Yanaihara N, Caplen N, Bowman E, et al: Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell. 9:189–198. 2006. View Article : Google Scholar

5 

Wijnhoven BP, Michael MZ and Watson DI: MicroRNAs and cancer. Br J Surg. 94:23–30. 2007. View Article : Google Scholar

6 

Luo HC, Zhang ZZ, Zhang X, Ning B, Guo JJ, Nie N, Liu B and Wu XL: MicroRNA expression signature in gastric cancer. Chin J Cancer Res. 21:74–80. 2009. View Article : Google Scholar

7 

Lu J, Getz G, Miska EA, et al: MicroRNA expression profiles classify human cancers. Nature. 435:834–838. 2005. View Article : Google Scholar : PubMed/NCBI

8 

Li Z, Lu J, Sun M, et al: Distinct microRNA expression profiles in acute myeloid leukemia with common translocations. Proc Natl Acad Sci USA. 105:15535–15540. 2008. View Article : Google Scholar

9 

Calin GA, Liu CG, Sevignani C, et al: MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA. 101:11755–11760. 2004. View Article : Google Scholar : PubMed/NCBI

10 

Volinia S, Calin GA, Liu CG, et al: A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA. 103:2257–2261. 2006. View Article : Google Scholar : PubMed/NCBI

11 

Zhang Z, Li Z, Gao C, et al: miR-21 plays a pivotal role in gastric cancer pathogenesis and progression. Lab Invest. 88:1358–1366. 2008. View Article : Google Scholar

12 

Iorio MV, Ferracin M, Liu CG, et al: MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 65:7065–7070. 2005. View Article : Google Scholar : PubMed/NCBI

13 

Takamizawa J, Konishi H, Yanagisawa K, et al: Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 64:3753–3756. 2004. View Article : Google Scholar : PubMed/NCBI

14 

Motoyama K, Inoue H, Nakamura Y, Uetake H, Sugihara K and Mori M: Clinical significance of high mobility group A2 in human gastric cancer and its relationship to let-7 microRNA family. Clin Cancer Res. 14:2334–2340. 2008. View Article : Google Scholar : PubMed/NCBI

15 

Varambally S, Cao Q, Mani RS, et al: Genomic loss of microRNA-101 leads to overexpression of histone methyltrans-ferase EZH2 in cancer. Science. 322:1695–1699. 2008. View Article : Google Scholar : PubMed/NCBI

16 

Cimmino A, Calin GA, Fabbri M, et al: miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA. 102:13944–13949. 2005. View Article : Google Scholar : PubMed/NCBI

17 

Costinean S, Zanesi N, Pekarsky Y, et al: Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci USA. 103:7024–7029. 2006. View Article : Google Scholar : PubMed/NCBI

18 

O’Donnell KA, Wentzel EA, Zeller KI, Dang CV and Mendell JT: c-Myc-regulated microRNAs modulate E2F1 expression. Nature. 435:839–843. 2005.PubMed/NCBI

19 

Murakami Y, Yasuda T, Saigo K, et al: Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene. 25:2537–2545. 2006. View Article : Google Scholar : PubMed/NCBI

20 

Tsai KW, Liao YL, Wu CW, et al: Aberrant hypermethylation of miR-9 genes in gastric cancer. Epigenetics. 6:1189–1197. 2011. View Article : Google Scholar : PubMed/NCBI

21 

Tanaka N, Toyooka S, Soh J, et al: Frequent methylation and oncogenic role of microRNA-34b/c in small-cell lung cancer. Lung Cancer. 76:32–38. 2012. View Article : Google Scholar : PubMed/NCBI

22 

Suh SO, Chen Y, Zaman MS, et al: MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer. Carcinogenesis. 32:772–778. 2011. View Article : Google Scholar : PubMed/NCBI

23 

Balaguer F, Link A, Lozano JJ, et al: Epigenetic silencing of miR-137 is an early event in colorectal carcinogenesis. Cancer Res. 70:6609–6618. 2010. View Article : Google Scholar : PubMed/NCBI

24 

Zhang Y, Wang X, Xu B, et al: Epigenetic silencing of miR-126 contributes to tumor invasion and angiogenesis in colorectal cancer. Oncol Rep. 30:1976–1984. 2013.PubMed/NCBI

25 

Schiffgen M, Schmidt DH, von Rücker A, Müller SC and Ellinger J: Epigenetic regulation of microRNA expression in renal cell carcinoma. Biochem Biophys Res Commun. 436:79–84. 2013. View Article : Google Scholar : PubMed/NCBI

26 

Michael MZ, O’ Connor SM, van Holst Pellekaan NG, Young GP and James RJ: Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res. 1:882–891. 2003.PubMed/NCBI

27 

Luo H, Zhang H, Zhang Z, et al: Downregulated miR-9 and miR-433 in human gastric carcinoma. J Exp Clin Cancer Res. 28:822009. View Article : Google Scholar

28 

Du Y, Liu Z, Gu L, et al: Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo. BMC Cancer. 12:2492012. View Article : Google Scholar

29 

Minor J, Wang X, Zhang F, et al: Methylation of microRNA-9 is a specific and sensitive biomarker for oral and oropharyngeal squamous cell carcinomas. Oral Oncol. 48:73–78. 2012. View Article : Google Scholar : PubMed/NCBI

30 

Tanaka T, Arai M, Wu S, et al: Epigenetic silencing of microRNA-373 plays an important role in regulating cell proliferation in colon cancer. Oncol Rep. 26:1329–1335. 2011.PubMed/NCBI

31 

Lujambio A, Calin GA, Villanueva A, et al: A microRNA DNA methylation signature for human cancer metastasis. Proc Natl Acad Sci USA. 105:13556–13561. 2008. View Article : Google Scholar : PubMed/NCBI

32 

Datta J, Kutay H, Nasser MW, et al: Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res. 68:5049–5058. 2008. View Article : Google Scholar : PubMed/NCBI

33 

Toyota M, Suzuki H, Sasaki Y, et al: Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer. Cancer Res. 68:4123–4132. 2008. View Article : Google Scholar : PubMed/NCBI

34 

Ando T, Yoshida T, Enomoto S, et al: DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: its possible involvement in the formation of epigenetic field defect. Int J Cancer. 124:2367–2374. 2009. View Article : Google Scholar : PubMed/NCBI

35 

Lehmann U, Hasemeier B, Christgen M, et al: Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer. J Pathol. 214:17–24. 2008. View Article : Google Scholar : PubMed/NCBI

36 

Grady WM, Parkin RK, Mitchell PS, et al: Epigenetic silencing of the intronic microRNA hsa-miR-342 and its host gene EVL in colorectal cancer. Oncogene. 27:3880–3888. 2008. View Article : Google Scholar : PubMed/NCBI

37 

Suzuki H, Maruyama R, Yamamoto E and Kai M: DNA methylation and microRNA dysregulation in cancer. Mol Oncol. 6:567–578. 2012. View Article : Google Scholar

38 

Guo LH, Li H, Wang F, Yu J and He JS: The tumor suppressor roles of miR-433 and miR-127 in gastric cancer. Int J Mol Sci. 14:14171–14184. 2013. View Article : Google Scholar : PubMed/NCBI

39 

Wada R, Akiyama Y, Hashimoto Y, Fukamachi H and Yuasa Y: miR-212 is downregulated and suppresses methyl-CpG-binding protein MeCP2 in human gastric cancer. Int J Cancer. 127:1106–1114. 2010. View Article : Google Scholar : PubMed/NCBI

40 

Hashimoto Y, Akiyama Y, Otsubo T, Shimada S and Yuasa Y: Involvement of epigenetically silenced microRNA-181c in gastric carcinogenesis. Carcinogenesis. 31:777–784. 2010. View Article : Google Scholar : PubMed/NCBI

41 

Shen R, Pan S, Qi S, Lin X and Cheng S: Epigenetic repression of microRNA-129-2 leads to overexpression of SOX4 in gastric cancer. Biochem Biophys Res Commun. 394:1047–1052. 2010. View Article : Google Scholar : PubMed/NCBI

42 

Suzuki H, Yamamoto E, Nojima M, et al: Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect. Carcinogenesis. 31:2066–2073. 2010. View Article : Google Scholar : PubMed/NCBI

43 

Rotkrua P, Akiyama Y, Hashimoto Y, Otsubo T and Yuasa Y: MiR-9 downregulates CDX2 expression in gastric cancer cells. Int J Cancer. 129:2611–2620. 2011. View Article : Google Scholar : PubMed/NCBI

44 

Tsai KW, Wu CW, Hu LY, et al: Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer. Int J Cancer. 129:2600–2610. 2011. View Article : Google Scholar : PubMed/NCBI

45 

Zhu A, Xia J, Zuo J, et al: MicroRNA-148a is silenced by hypermethylation and interacts with DNA methyltransferase 1 in gastric cancer. Med Oncol. 29:2701–2709. 2012. View Article : Google Scholar : PubMed/NCBI

46 

Li P, Chen X, Su L, et al: Epigenetic silencing of miR-338-3p contributes to tumorigenicity in gastric cancer by targeting SSX2IP. PloS One. 8:e667822013. View Article : Google Scholar : PubMed/NCBI

47 

Lei H, Zou D, Li Z, et al: MicroRNA-219-2-3p functions as a tumor suppressor in gastric cancer and is regulated by DNA methylation. PLoS One. 8:e603692013. View Article : Google Scholar : PubMed/NCBI

48 

Deng H, Guo Y, Song H, et al: MicroRNA-195 and microRNA-378 mediate tumor growth suppression by epigenetical regulation in gastric cancer. Gene. 518:351–359. 2013. View Article : Google Scholar : PubMed/NCBI

49 

Li Y, Li B, Zhang Y, Xiang CP, Li YY and Wu XL: Serial observations on an orthotopic gastric cancer model constructed using improved implantation technique. World J Gastroenterol. 17:1442–1447. 2011. View Article : Google Scholar : PubMed/NCBI

50 

Momparler RL: Pharmacology of 5-Aza-2′-deoxycytidine (decitabine). Semin Hematol. 42(Suppl 2): S9–S16. 2005.

51 

Takai D and Jones PA: The CpG island searcher: a new WWW resource. In silico Biol. 3:235–240. 2003.PubMed/NCBI

52 

Chen Q, Chen X, Zhang M, Fan Q, Luo S and Cao X: miR-137 is frequently down-regulated in gastric cancer and is a negative regulator of Cdc42. Dig Dis Sci. 56:2009–2016. 2011. View Article : Google Scholar : PubMed/NCBI

53 

Yamamoto E, Suzuki H, Maruyama R and Shinomura Y: Developing technologies for epigenomic analysis and clinical application of molecular diagnosis. Rinsho Byori. 60:637–643. 2012.(In Japanese).

54 

Langevin SM, Stone RA, Bunker CH, et al: MicroRNA-137 promoter methylation is associated with poorer overall survival in patients with squamous cell carcinoma of the head and neck. Cancer. 117:1454–1462. 2011. View Article : Google Scholar : PubMed/NCBI

55 

Furuta M, Kozaki KI, Tanaka S, Arii S, Imoto I and Inazawa J: miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma. Carcinogenesis. 31:766–776. 2010. View Article : Google Scholar : PubMed/NCBI

56 

Li Y, Kong D, Ahmad A, Bao B, Dyson G and Sarkar FH: Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion. Epigenetics. 7:940–949. 2012. View Article : Google Scholar : PubMed/NCBI

57 

Momparler RL: Epigenetic therapy of cancer with 5-aza-2′-deoxy-cytidine (decitabine). Semin Oncol. 32:443–451. 2005.

Related Articles

Journal Cover

December-2014
Volume 45 Issue 6

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Li Y, Xu Z, Li B, Zhang Z, Luo H, Wang Y, Lu Z and Wu X: Epigenetic silencing of miRNA‑9 is correlated with promoter‑proximal CpG island hypermethylation in gastric cancer in vitro and in vivo. Int J Oncol 45: 2576-2586, 2014
APA
Li, Y., Xu, Z., Li, B., Zhang, Z., Luo, H., Wang, Y. ... Wu, X. (2014). Epigenetic silencing of miRNA‑9 is correlated with promoter‑proximal CpG island hypermethylation in gastric cancer in vitro and in vivo. International Journal of Oncology, 45, 2576-2586. https://doi.org/10.3892/ijo.2014.2667
MLA
Li, Y., Xu, Z., Li, B., Zhang, Z., Luo, H., Wang, Y., Lu, Z., Wu, X."Epigenetic silencing of miRNA‑9 is correlated with promoter‑proximal CpG island hypermethylation in gastric cancer in vitro and in vivo". International Journal of Oncology 45.6 (2014): 2576-2586.
Chicago
Li, Y., Xu, Z., Li, B., Zhang, Z., Luo, H., Wang, Y., Lu, Z., Wu, X."Epigenetic silencing of miRNA‑9 is correlated with promoter‑proximal CpG island hypermethylation in gastric cancer in vitro and in vivo". International Journal of Oncology 45, no. 6 (2014): 2576-2586. https://doi.org/10.3892/ijo.2014.2667