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. 2011 Oct 1;6(10):1189–1197. doi: 10.4161/epi.6.10.16535

Aberrant hypermethylation of miR-9 genes in gastric cancer

Kuo-Wang Tsai 1, Yu-Lun Liao 2, Chew-Wun Wu 3, Ling-Yueh Hu 2, Sung-Chou Li 2,4, Wen-Ching Chan 2,4, Meng-Ru Ho 2,4, Chun-Hung Lai 2, Hsiao-Wei Kao 2, Wen-Liang Fang 3,5, Kuo-Hung Huang 3,5, Wen-chang Lin 3,4,✉
PMCID: PMC3225840  PMID: 21931274

Abstract

Carcinogenesis of the stomach involves multiple steps including genetic mutation or epigenetic alteration of tumor suppressor genes or oncogenes. Recently, tumor suppressive miRNAs have been shown to be deregulated by aberrant hypermethylation during gastric cancer progression. In this study, we demonstrate that three independent genetic loci encoding for miR-9 (miR-9-1, miR-9-2 and miR-9-3) are simultaneously modified by DNA methylation in gastric cancer cells. Methylation-mediated silencing of these three miR-9 genes can be reactivated in gastric cancer cells through 5-Aza-dC treatment. Subsequent analysis of the expression levels of miR-9 showed that it was significantly downregulated in gastric cancers compared with adjacent normal tissues (p value < 0.005). A similar tendency toward a tumor-specific DNA methylation pattern was shown for miR-9-1, miR-9-2 and miR-9-3 in 72 primary human gastric cancer specimens. Ectopic expression of miR-9 inhibited cell proliferation, migration and invasion, suggesting its tumor suppressive potential in gastric cancer progression.

Key words: miR-9, DNA methylation, CpG islands, gastric cancer

Introduction

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of their target mRNAs through translational repression or mRNA cleavage.1 Dysregulation of miRNAs is assumed to play an important role in cancer development by regulating cell proliferation, differentiation, apoptosis and carcinogenesis.2–4 Previous studies have shown that many miRNAs are aberrantly overexpressed or downregulated during gastric cancer progression, including miR-16, miR-21, miR-101, miR-126, miR-129, miR-181c, miR-196b and −200.5–12 These miRNAs could play oncogenic or tumor-suppressive functions in the regulation of cell growth, apoptosis and cell migration by repressing their target genes.

Various molecular mechanisms lead to miRNA dysregulation, such as genetic mutation, epigenetic aberration and deregulated transcriptional activity. Among them, epigenetic regulation could significantly alter gene expression through histone modification and DNA methylation, which are important in the regulation of cell growth and development in mammals. Previous studies have demonstrated that several tumor suppressor genes are silenced in gastric carcinogenesis with abnormal promoter region hypermethylations.13 Similarly, several tumor-suppressive miRNAs have been shown to acquire hypermethylation patterns at their promoter regions in human gastric cancers, including miR-34b/c, miR-129, miR-137, miR-181C, miR-199a, miR-212, miR-512 and miR-516.6,9,14–21

miR-9 is a highly conserved miRNA found in insects and primates.22 In humans, there are three independent miR-9 gene loci, miR-9-1, miR-9-2 and miR-9-3, which are located at chromosomes 1, 5 and 15, respectively. According to the miRBase definition, these three loci encode the same mature miR-9 sequences, while the hairpin precursors are varied. Dysregulation of miR-9 expression has profound effects on physiology and pathology, including cancer, neuronal differentiation and myocardial hypertrophy.23–30 Several lines of evidence have indicated that miR-9 can function as a tumor suppressor in various cancers.23,24,27,29,31–33 In gastric cancer, miR-9 is under-expressed and ectopic expression of miR-9 can influence cell growth and the cell cycle.24,29,32,33 However, there is no study examining the mechanisms involved in the differential regulation of all three miR-9 gene loci. In this study, we explore the promoter methylation status of the miR-9-1, miR-9-2 and miR-9-3 gene loci in gastric cancer tissues.

Results

Epigenetic regulation of miR-9 expression in human gastric cancer cell lines.

We previously performed a miRNA profile scan for human AGS gastric cancer cells following the treatment of DNA demethylation agents and identified several methylation-associated miRNAs, including miR-9.9 We now demonstrated that the expression levels of mature miR-9 were modulated through DNA demethylation treatment at various time periods (0–4 days after treatment) in AGS cells (Fig. 1A). To study the biological roles of miR-9 DNA methylation in gastric cancer cells, we examined the expression levels of miR-9 in the presence or absence of 5-Aza-dC. Our results show that the expression level of miR-9 was reduced in AGS and HR cells; miR-9 expression in these cells could be restored when genomic DNA was hypomethylated (Fig. 1B and C). This suggests that the transcriptional activity of mature miR-9 is tightly regulated and can be silenced by DNA methylation in AGS and HR gastric cancer cells.

Figure 1.

Figure 1

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Expression of miR-9 is epigenetically repressed in gastric cancer cells. (A) Expression levels of miR-9 in cells treated with 5-Aza-dC were detected using the stem-loop qRT-PCR method at various time periods (0–4 days). (B) Expression levels of miR-9 were examined in five human gastric cancer cells. The PCR products were analyzed on a 3% NuSieve/l% agarose gel. U6 snRNA was used as internal control. (C) Real-time PCR analysis of miR-9 in human cell lines before and after demethylation. U6 expression was used as internal control, and gene expression was calculated relative to the internal control (ΔCt). The relative expression of miR-9 was calculated using the standard equation 10,000x (2−ΔCt).

The transcriptional activity of three miR-9 genes is regulated by DNA methylation.

Expression of the human mature miR-9 originates from three genomic loci in chromosomes 1, 5 and 15. All three loci could independently contribute to the expression of miR-9. There is no current report on the simultaneous modulation of the three miR-9 loci. Interested in learning more about the epigenetic modifications in the three miR-9 loci, we searched the UCSC database and found three CpG-rich regions located at the putative transcription start sites of miR-9-1, miR-9-2 and miR-9-3 loci, which imply that their transcriptional activity may indeed be controlled by DNA methylation. We sought to determine whether these three miR-9 genes loci were modified via CpG methylation using molecular biology approaches. We analyzed the methylation status of three CpG-rich regions in five human gastric cancer cell lines using a COBRA approach (Fig. 2). We observed completely hypermethylated CpG island upstream of miR-9-1 in five human gastric cancer cells (Fig. 2B). High frequency of DNA methylation was observed in the promoter regions of miR-9-2 and miR-9-3 loci (Fig. 2B). Subsequent bisulfite sequencing data of miR-9-1, miR-9-2 and miR-9-3 CpG-rich regions in AGS and TSGH was consistent with the COBRA results (Fig. S1). Therefore, epigenetic modulation by DNA methylation could play a significant role in the expression of all three miR-9 loci. This is further supported by demethylation treatment, which reactivated the transcriptional activities of pri-miR-9-1, miR-9-2 and miR-9-3 in all examined gastric cancer cell lines (Fig. 2C). Therefore, the three human miR-9 genes could be epigenetically regulated via DNA methylation in gastric cancer cells.

Figure 2.

Figure 2

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Three miR-9 genes are silenced by DNA methylation. (A) Schematic representation of the locations of the three miR-9 genes (miR-9-1, miR-9-2 and miR-9-3). EST transcripts were identified using the UCSC website (http://genome.ucsc.edu/). Stem-loops indicate the positions of individual miRNAs, and green blocks indicate their neighboring CpG-rich regions. Red asterisk indicates CpG islands. (B) COBRA was used to analyze the methylation status of each CpG island from the three miR-9 genes in human gastric cancer cell lines. Arrows indicate the unmethylated (u)/methylated (m) alleles. (C) Expression of the three miR-9 primary transcripts is reactivated with 5-Aza-dC treatment in five human gastric cancer cell lines. The methylation status measurement of the individual CpG-rich regions was duplicated using the COBRA assay. M, M/U and U indicate that the CpG-rich region contains methylated CpGs only, both methylated and unmethylated CpGs, or unmethylated CpGs only, respectively.

Tumor-specific DNA methylation suppresses the expression of miR-9 in gastric cancer tissues.

We examined the expression of miR-9 in 72 pairs of gastric cancer specimens. A decreased expression level of miR-9 was considerably noted in 80.6% of the tumor tissues examined (58 of 72 cases). The expression levels of miR-9 were significantly lower in tumors than in their corresponding normal-tissue counterparts (p value < 0.005) (Fig. 3). We further investigated whether the tumor-specific methylation resulted in miR-9 downregulation in gastric cancers. Furthermore, we explicitly analyzed the methylation status of individual CpG islands of all three independent miR-9 gene loci in the 72 gastric cancer samples using the COBRA approach. As shown in Figure 4, three CpG islands exhibited a tendency towards hypermethylation in gastric cancer genomic DNA. Subsequent bisulfite sequencing data of miR-9-1, miR-9-2 and miR-9-3 CpG-rich regions in selected patients were consistent with the COBRA results (Fig. 4D). Tumor-specific DNA methylation patterns of the three genes were found in 33 of 72 (45.8%), 45 of 72 (62.5%) and 44 of 72 (61.1%) of the primary gastric cancers analyzed, respectively (Fig. 5). Interestingly, we observed a similar methylation pattern of three CpG islands in most of the gastric cancer samples examined. The expression level of miR-9 was significantly reduced in primary gastric cancer cases versus adjacent normal tissues, which frequently contains tumor-specific methylation in three independent pri-miR-9 genes loci (46.6%, 27 of 58) (Fig. 5). These results indicate that dysregulation of hypermethylation concurrently regulates the three pri-miR-9 paralogous loci, which dramatically reduces expression of miR-9 in the gastric cancer tissue samples. We also analyzed the correlation between the methylation status of miR-9 genes and the clinicopathological features of patients with primary gastric cancer. We observed that hypermethylation of miR-9-3 was associated with poor clinical outcomes including lymphovascular involvement and reduced survival rate (Table 1).

Figure 3.

Figure 3

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Expression levels of miR-9 were examined in 72 gastric cancer patients. Expression levels of miR-9 in the gastric cancer tissues from 72 patients were examined using quantitative stem-loop PCR. U6 was used as internal control. All samples were assessed in triplicate and analyzed using a Student's t test (p < 0.05 was considered significant).

Figure 4.

Figure 4

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Methylation status of the three miR-9 genes in genomic DNA from 72 gastric cancer samples. (A–C) Methylation status of each CpG island at miR-9-1, miR-9-2 and miR-9-3 was analyzed in primary gastric cancer samples using COBRA. Asterisks indicate that tumor-specific methylation was detected. (D) Methylation status of one case was examined by bisulfite sequencing. Each row represents a single clone for each individual genomic DNA. Open and filled squares represent unmethylated and methylated CpG sites, respectively.

Figure 5.

Figure 5

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Correlation between DNA methylation status and miR-9 expression in tumors from 72 gastric cancer patients. Expression levels of miR-9 in gastric cancer tissues from 72 patients, duplicated from Figure 3. Tumor-specific methylation of miR-9-1, miR-9-2 and miR-9-3 are indicated by triangle, circle and diamond, respectively. Asterisks indicate simultaneous tumor-specific methylation in the three miR-9 genes.

Table 1.

Clinicopathological relationship in GCA patients with methylation status of pri-mir-9-3

Variables Uumethylation (n = 12) Methylation (n = 57) p
Age (years) means (±SD) 63.9 ± 14.2 66.3 ± 13.3 0.588
Age <65 yrs 6 21 0.396
≥65 yrs 6 36
Sex Male 8 45 0.453
Female 4 12
Tumor size <4 cm 1 4 0.606
4–8 cm 6 36
>8 cm 5 17
Tumor location 0.781
Upper stomach 3 16
Middle stomach 1 9
Lower stomach 7 30
Whole stomach 1 2
Cell grade 0.312
Poor differentiated 10 36
Moderate differentiated 2 21
Well differentiated - -
Gross appearance 0.440
Borrmann 0 - -
Borrmann I, II 1 14
Borrmann III, IV 11 43
Cancer-stroma relationship 0.033*
Medullary type 0 5
Intermediate type 5 41
Scirrhous type 7 11
Tumor infiltrating patterns 0.315
INF (infiltrating)α 0 3
INFβ 4 30
INFγ 8 24
Lauren'fs histological classification 0.151
Intestinal type 4 32
Diffuse type 8 25
Nodal involvements. 0.340
Negative 0 7
Positive 12 50
Lymphovascular involvements 0.017*
Negative 6 9
Positive 6 48
Depth of cancer invasion 1.000
m, 0 1
sm 6 25
mp 6 27
se, sei 0 4
TNM Stage 0.238
I 0 4
II 2 17
III 7 15
IV 3 21
Overall Survival rate (5-year) 20.0% 34.9% 0.015*
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Aberrant expression of miR-9 is involved in cancer cell migration.

According to these observations, we hypothesized that miR-9 could play a tumor suppressive role in gastric cancer progression. To address this, we performed analysis of cell proliferation and motility after transfection of pri-miR-9 (or control) in AGS and HR cells (Fig. 6). As shown in Figure 6B, ectopic expression of miR-9 in HR cells significantly suppressed cell proliferation. Contrarily, high expression level of miR-9 did not cause any alteration of AGS cells' growth, but it could lead to decreasing expression levels of MMP2, MMP9, Twist and N-Cadherin (data not shown). We therefore used a migration and invasive assay in gastric cells with/without miR-9 overexpression. Our data showed that miR-9 led to the suppression of migration and invasiveness of AGS and HR cells in vitro (Fig. 6C). Therefore, we concluded that miR-9 overexpression in gastric cancer cells resulted in the suppression of gastric cancer cell motility, invasiveness and proliferation.

Figure 6.

Figure 6

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Ectopically overexpressed miR-9 suppressed cell motility. (A) Overexpression of the miR-9 through lentivirus infection in AGS and HR was examined by stem-loop RT-PCR. Cells were infected with either control shLuc or miR-9 lentivirus for 24 h, which was followed by puromycin (2 µg/ml) selection. (B) Cell proliferation was performed using MTT assay. (C) Overexpression of miR-9 silenced cell migration and invasion activity in AGS (left part) and HR (right part) cells. Cell migration and invasiveness were assessed as described in Materials and Methods. For every filter, 30 fields were counted. Data are an average of triplicates for each condition. **p value < 0.001.

Discussion

DNA methylation is involved in silencing gene expression by establishing and maintaining a repressive status at gene promoters. Previous studies have demonstrated that abnormal methylation patterns lead to tumor formation through hypermethylated promoters of tumor suppressor genes.13 The basic transcription mechanism of miRNA is fundamentally similar to that of classical protein-coding genes and aberrant DNA hypermethylation had been shown to silence tumor-suppressive miRNAs in cancers. Recent studies have indicated that several miRNAs, including miR-34b/c, miR-129, miR-137, miR-181C, miR-199a, miR-212, miR-512 and miR-516, were dysregulated and showed aberrant methylation patterns in gastric cancer cells.6,9,14–21 The three pri-miR-9 transcripts are all located within non-protein coding transcripts (Fig. 2A). These novel transcripts may be primary transcripts for miR-9-1, miR-9-2 and miR-9-3 or perform other important function. In this study, we reported that the three independent gene loci contribute to mature miR-9 expression, which was silenced by DNA hypermethylation in gastric cancers (Fig. 5). Previous studies have shown that high frequency of hypermethylated CpG islands at miR-9-1 and miR-9-3 genes resulted in downregulation of miR-9 in human cancers, including breast cancer, colorectal cancer and renal cell carcinoma.23,24,27,31 However, miR-9-2 methylation-associated silencing of transcription was rarely mentioned. Trankenschuh et al. observed hypermethylation of miR-9-1 and miR-9-2 in hepatocellular carcinoma. Our study showed that DNA methylation occurs at three miR-9 genes, resulting in the low expression of miR-9 in gastric cancers. Hypermethylation of pri-miR-9-3 CpG islands was significantly associated with the poor clinical features of gastric cancer patients, which implied that miR-9 plays a tumor suppressive function in gastric cancer.

In this study, ectopic expression of miR-9 could suppress gastric cancer cell migration and proliferation (Fig. 6). Similar findings indicate that miR-9 plays a tumor-suppressive role via repressing tumor-associated genes (NFκB1 and RAB34) in gastric cancer.29,33 Significantly, in human cancers, miR-9 expression levels or methylation status are associated with several clinicopathological features, such as tumor grade, metastatic status and survival rate.23,27,31 Contrary to the above results, it has been showed that miR-9 is significantly deregulated in breast cancer or colorectal cancer, with increasing cancer migration, through repression of α-cadherin or E-cadherin expression.28,35 Taken together, these data suggest that miR-9 plays a dual role, depending on its target genes and the cancer type.

In this study, tightly repressed expression of the three primary miR-9 transcripts in gastric cancer cell lines could be restored through demethylating agent treatment (Fig. 2). We also observed high frequency of hypermethylation at miR-9-1, miR-9-2 and miR-9-3 loci in gastric cancers cells (Fig. 4). Analyzing the methylation status of all three human miR-9 gene loci in gastric cancers in detail, we found that DNA methylation tightly repressed miR-9 through the simultaneous methylation of the CpG-rich regions of three independent genes.

Materials and Methods

Cell lines and 5-Aza-dC treatment.

Five human gastric cancer cell lines (AGS, AZ-521, HR, NUGC and TSGH) were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium supplemented with 10% inactivated FBS (Invitrogen, Carlsbad, CA USA). Gastric cells were cultured in the presence or absence of 2.5 µM 5-Aza-dC for various time periods (0–4 days after treatment). Then, total RNA was prepared using TRIZOL (Invitrogen, Carlsbad, CA USA), according to the manufacturer's protocol.

Stem-loop RT-PCR.

Next, 1 ug of total RNA was reverse-transcribed with a stem-loop RT reaction with miR-9 RT primers and SuperScript III Reverse Transcriptase according to the user's manual (Invitrogen, Carlsbad, CA USA). The reaction was performed with the following incubation conditions: 30 min at 16°C, followed by (20°C/30 s, 42°C/30 s, 50°C/1 s) for 50 cycles. The enzyme was subsequently inactivated by incubation at 85°C for 5 min. Gene expression was detected using a SYBR Green I assay (Applied Biosystems, Foster City, CA USA), and the expression levels of miR-9 were normalized to that of U6 (ΔCt = target microRNA Ct-U6 Ct).36 To simplify the presentation of the data, the relative expression values were multiplied by 105.37 The primary transcripts of miR-9-1, miR-9-2 and miR-9-3 were examined by RT-PCR with a gene-specific primer. The individual primers used in this study follow:

miR-9-RT: 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG TCA TAC AG-3′

miR-9-GSF: 5′-CGG CGG TCT TTG GTT ATC TAG C-3′

pri-miR-9-1-F: 5′-GAG GCT GCG TGG AAG AGG-3′

pri-miR-9-1-R: 5′-ACT CCA CAC CAC TCA TAC AGC-3′

pri-miR-9-2-F: 5′-TGG CAA GAG GAA GAC AGA GG-3′

pri-miR-9-2-R: 5′-TGA AGA CCA ATA CAC TCA TAC AGC-3′

pri-miR-9-3-F: 5′-GAG GCC CGT TTC TCT CTT TG-3′

pri-miR-9-3-R: 5′-AGC TTT ATG ACG GCT CTG TG-3′

Assays of CpG island methylation.

DNA bisulfite conversion. Genomic DNA was extracted from cultured cells or gastric carcinoma tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA USA), and an aliquot (2 µg) was then subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research Corporation, Orange, CA USA). The bisulfite conversion reaction was incubated at 98°C for 10 min and then at 64°C for 2.5 h, with a final incubation at 4°C for up to 20 h in a PCR thermocycler.

COBRA and bisulfite sequencing analysis. The bisulfite-converted genomic DNA was used for the methylation analysis of miR-9-1, miR-9-2 and miR-9-3 with the specific methylation primers. The PCR conditions were as follows: 94°C for 10 min, followed by 35 cycles of 94°C/1 min, 60°C/1 min and 72°C/30 s, with a final extension at 72°C for 10 min using a PCR thermocycler and HotStart Taq DNA polymerase (Qiagen, Hilden, Germany). The methylation status of the genomic DNA of individual samples was also examined by BstuI or TaqI digestion (New England Biolabs, MA). The digested PCR fragments were then separated on 2% agarose gel. After a PCR reaction was induced, PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI USA), and several clones were randomly selected for sequencing. The methylation primers used in this study follow:

miR-9-1-mF: 5′-GAT TTA GGT AGA GGT TTT TTT AgT TT-3′ (248 bp)

miR-9-1-mR: 5′-TTA ACT ACC CAT TTC CCC TTT TAA T-3′

miR-9-2-mF: 5′-ATG TTA GGA AAA TAA AGT TGG GGT T-3′ (181 bp)

miR-9-2-mR: 5′-CCC CTC CTC TAA AAC AAC TAA AAA-3′

miR-9-3-mF: 5′-GTT TGT TTA TTT TTT TTG GTT TTT-3′ (220 bp)

miR-9-3-mR: 5′-AAA TTA TAA AAA TCA TTT CTA CTT TC-3′

Clinical samples and statistical analysis.

In this study, 72 human gastric carcinoma tissues and paired normal adjacent tissues were obtained from the Department of Surgery, Veterans General Hospital, Taipei, Taiwan. Expression levels of miR-9 in all gastric tissues were performed by stem-loop qRT-PCR. All reactions were run in triplicate and analyzed by t-test. For internal control, U6 expression was used and expression level of miR-9 was indicated as ΔCt (ΔCt = target microRNA Ct-U6 Ct). The difference was considered to be significant when the p-value was less than 0.05.

Cell proliferation, cell migration assay and invasion assay.

For cell proliferation analysis, 5,000 living cells were plated onto 96-well plates. Cell growth was determined at 0–4 days using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; sigma). Cells were tested for migration and invasion abilities in vitro in transwell chambers (Costar, Lowell, USA). The lower side or the upper side of the polycarbonate membranes (containing 8-µm pores) of the transwell were coated with 50 µg/ml of type I collagen or 80 µg per well of Matrigel and were used for migration or invasion assays, respectively. Cells were added to the upper chamber of a transwell. After incubation for 24 h at 37°C, the cells at the lower side were prepared for Giemsa stain. The level of migration or invasion was determined using a microscope at 200x magnification. All experiments were repeated three times.

Acknowledgments

This study was supported in part by research funding from National Sciences Council (NSC 98-2311-B-001-016, DOH100-TD-C-111-007) and Kaohsiung Veterans General Hospital (VGHKS 100-122).

Abbreviations

miRNAs

microRNAs

COBRA

combined bisulfite restriction analysis

5-Aza-dC

5-Aza-deoxycytidine

pri-miRNA

primary microRNA

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplementary Material

Supplementary Material
epi0610_1189SD1.pdf (1.1MB, pdf)

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