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Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo
© Du et al.; licensee BioMed Central Ltd. 2012
Received: 10 January 2012
Accepted: 4 May 2012
Published: 15 June 2012
Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression.
Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed.
Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02).
In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may significantly down-regulate their transcription regularly.
miRNA are an abundant class of small non-coding RNAs that mainly regulate gene expression at the post-transcriptional level. They play critical roles in the renewal and differentiation of stem cells and help maintain cell lineages. Previous research has shown that in cancer several of the miR genes such as miR-200b/200a/429, miR-21, miR-30b, miR-30d, miR-31, and miR-423 are upregulated, while other miR genes such as miR-143 and miR-145 are downregulated [1–3]. Evidence suggests that changes in miRNA expression occur frequently in many cancers and these variations either contribute to carcinogenesis or reflect the development and progression of cancers.
There are a number of pathways that may affect mature miRNA levels in cells and tissues, such as gene amplification or deletion, transcriptional upregulation or downregulation, post-transcriptional processing, and miRNA degradation [4–7]. It is well known that some intragenic miR genes, such as miR-218-2, are coordinately transcribed with their host genes through co-regulation mechanisms . However, many miR genes are extragenic and a certain proportion of intragenic miR genes such as miR-9-1 are transcribed in a host gene-independent pattern . Because the exact promoter region of most miR genes are not characterized, especially with regard to the extragenic miR genes, the exact regulatory mechanisms of miR transcription are far from clear.
Methylation or hypermethylation of CpG islands in the region of transcription starting sites (TSS) is generally recognized to repress gene transcription epigenetically. Unlike protein-coding genes that may span multiple CpG islands, the miR genes may be shorter than a CpG island, and in some cases, multiple miR genes (i.e., a miR gene cluster) may be located within or flanking a single CpG island (Additional file 1: Table S1). Aberrant methylation of CpG islands associated with miR genes, such as let-7a-3 and miR-34a, is frequently observed in many cancers [10, 11]. It has been suggested that methylation of the CpG islands that are associated with miR genes (i.e. miR-203, miR-152, miR-124-1, miR-34b/c, miR-129-2, miR-9-1, miR-130b, miR-124-2, and miR-181c) might inversely correlate with their expression levels [12–17]. However, whether or not transcription of miR genes is regularly affected by the methylation status of miR CpG islands has not been systemically studied.
It is well known that abnormal methylation or demethylation of CpG islands in a small proportion (<1%) of the cell population can be sensitively detected in cellular heterozygous tissue samples. This demonstrates the advantage of methylation analysis over alterations of gene expression at the RNA and protein levels that can only be detected when such a changes is present in a large proportion of a cell population in a sample . Our bioinformatic analysis shows 50, 9, and 70 of 721 human miR genes in the miRbase (Release 14.0) are located, respectively, within, flanking, and near CpG islands (collectively we will refer to these as miR CpG islands; Additional file 1: Table S1). We hypothesize that aberrant methylation of miR CpG islands may occur during development and progression of cancers. Therefore they could be used as candidate genes not only for prediction of cancer prognosis, but also for investigation of the methylation-expression association in vivo. Thus, CpG islands of 9 disease-related miR genes, including 5 extragenic miR genes or gene clusters (miR-9-3, miR-137, miR-200b/200a/429, miR-203, and miR-375) and 4 intragenic genes or gene clusters (miR-9-1, miR-34b/c, miR-193b/365-1, and miR-210), were selected as the representative genes in the present study (Additional file 1: Table S1). The methylation-expression association for 3 of these miR genes has not been previously established (Additional file 1: Table S2) [12, 14, 19–27]. We initially screened for gastric carcinoma (GC)- or host-related aberrant miR methylation and then investigated the methylation-expression association in vitro and in vivo. Associations between clinicopathological features of GC patients and methylation of these miR CpG islands were also analyzed.
Cell line sources and cell culture
Source information of used cell lines used in this study: RKO cell line, provided by Dr. Guoren Deng at University of California San Francisco; SW480 and HCT116 were provided by Dr. Yuanjia Chen at Peking Union Medical College Hospital; MKN74 and 293 T provided by Tokyo Medical and Dental University; PC-3 was purchased from the Cell Line Bank at the Chinese Academy of Medical Science; HL60 and KG1A were obtained from the Hematology Department of Peking University First Hospital; Du145 was obtained from Hanmi Pharmacy Company; Siha was provided by Peking University People’s Hospital. HepG2 was provided by Dr. Qingyun Zhang, Calu3 and A549 by Dr. Zhiqian Zhang, H1299 and AGS by Dr. Chengchao Shou, MKN45 by Dr Youyong Lv, and other cell lines (SGC7901, BGC823, MGC803, HeLa and GES-1) by Dr. Yang Ke, all at Peking University Cancer Hospital/Institute. These cell lines were cultured at 37°C in 5% CO2, using various culture media. MKN45, MKN74, SGC7901, BGC823, MGC803, HL60, KG1A, A549, H1299, GES-1, HepG2, 293 T, Du145, and RKO were cultured in 90% RPMI-1640 and 10% FBS. PC-3 and AGS were cultured in 90% F-12 and 10% FBS. Calu3, HeLa and Siha were cultured in 90% DMEM and 10% FBS. SW480 and HCT116 were cultured in 90% DMEM:RPMI-1640 (1:1) and 10% FBS.
Patients and tissue samples
Surgical primary GC samples and their paired non-cancerous surgical margin (SM) samples were collected from 112 inpatients (average age 59.2 years [range, 32–79]; 80 males and 32 females; 78 non-cardiac GCs and 34 cardiac GCs; 40 GCs at pTNM stage I ~ II and 59 at the stage III ~ IV) at Peking University Cancer Hospital. Follow-up data for all patients was collected for at least five years. All clinical samples, as well as histopathological and followup information for each case were obtained according to approved institutional guidelines. Gastric biopsies from 13 healthy subjects and 38 gastritis outpatients collected from the same hospital were used as non-cancer patient controls. Before bisulfite modification each patient’s gastric genomic DNA sample was analyzed for the presence of H. pylori-specific 23 S rDNA by a PCR assay as described previously . The Institutional Review Boards of Peking University Cancer Hospital and Institute approved the study (#2011041207), and all patients gave written informed consent.
DNA extraction and bisulfite modification
Cancer cell line and tissue sample genomic DNA (1.8 μg) was isolated using phenol/chloroform extraction . The unmethylated cytosine residues in the DNA samples were converted to uracil residues (becoming thymidine residues in PCR products) by the addition of 5 M sodium bisulfite . The Wizard® DNA Clean-Up System Kit (Promega) was used to purify the bisulfite-treated DNA before PCR amplification.
PCR amplification and quantification of miRCpG island methylation by DHPLC
Fresh PCR products of miR CpG islands amplified with the CpG-free universal primer sets were cloned with the pGEM-T Easy kit (Promega, Madison, USA) and sequenced with an Applied Biosystems 3730xl DNA Analyzer at SinoGeneMox Company (Beijing, China).
Extraction of RNA and detection of mature miRNA level with quantitative RT-PCR assays
Total 50 ng RNA was extracted from fresh tissue samples or cell lines using the TRIzol reagent (Life Technologies, Carlsbad, USA) according to the manufacturer’s protocol. Corresponding cDNA samples were synthesized using the TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies) with miR-specific stem-loop inverse transcription (RT) primers (specific for miR-375 #RT000564, miR-34b #RT000427, miR-137 #RT000593, and miR-9 #RT000583). The RT conditions used were 16°C for 30 min ➔ 42°C for 30 min ➔ 85°C for 5 min. The miRNA levels were then analyzed using a TaqMan Gene Expression Master Mix kit (Life Technologies) with the corresponding probe and primers (Life Technologies, miR-375 #TM000564, miR-34b #TM000427, miR-137 #TM000593, and miR-9 #TM000583). U6 (Life Technologies, #RT001093 and #TM001093) was used as the internal reference. The PCR cycling conditions were 95°C for 10min ➔ followed by 40 cycles of 95°C for 20 sec ➔ 60°C for 1 min. Expression levels of miR-200b and miR-210 were determined using a standard polyA RT-PCR assay. Sequences of the RT adaptor primer, the universal inverse primer and the U6 primer in the regular polyA RT-PCR assay are shown in Additional file 1: Table S5. RT conditions were 55°C for 5 min ➔ followed by 25°C for 10 min ➔ 42°C for 1 hr ➔ and 70°C for 5 min. The PCR cycling conditions were 95°C for10min ➔ followed by 40 cycles of 95°C for 20 sec ➔ and 61°C for 1 min.
SPSS 16.0 Trend-test and Pearson’s Chi-square test were used to analyze the miR methylation frequency difference between normal biopsies, gastritis lesions, GC and SM samples. Kruskal-Wallis H-test and One-Way ANOVA were used to analyze the miR methylation proportion differences between normal biopsies, gastritis lesions, GC, and SM samples. Fisher’s exact test, Pearson’s Chi-square test, and Trend-test were used to analyze the association between miR methylation positive rates and the clinicopathological features. The Mann–Whitney U-test and Student’s t-test were used to analyze the association between the proportion of methylated miR alleles and the clinicopathological features. Kaplan-Meier and Cox-Proportional Hazards methods were used for univariate and multivariate analysis to compare overall survival of GC patients with differences in methylation status of miR CpG islands. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.
Characterization of methylation or demethylation of 6 miRCpG islands related to the development of GCs
Methylation status of miR CpG islands in gastric mucosa samples with different pathological changes in GC and non-cancerous control patients
Group of gastric samples
Proportion (%) of methylated miRin the miRmethylation-positive samples
Positive rate (%)
χ 2 -value
Median[25% ~ 75%]
t/F/χ 2 -value
20 ± 4
t = −3.039
32 ± 4
43 ± 6
F = 1.639
42 ± 2
36 ± 2
36 ± 2
χ 2 = 8.065
21 ± 3g
37 ± 4
χ 2 = 0.658
χ 2 = 17.883
52 [46–100] e
χ 2 = 2.957
3 [3–4] e
8/28 (28.6) c
Except for miR-9-1 and miR-137, the methylation positive rates or proportions of methylated miR-9-3, miR-34b, miR-210, and miR-200b in GCs were similar to those in SMs (Table 1). To validate if methylation of some of these miR genes is a field-effect happens simultaneously in both cancerous and non-cancerous tissues in the stomach due to the same exposure to environmental factors, we detected the methylation data in another subset of GC and SM samples from 84 patients and found that the positive rate and proportion of methylated miR-9-1 in GCs was still significantly higher than that in SMs (60.7% versus 32.1%; Pearson’s Chi-square test, P = 0.001; Sign rank test, P < 0.001; Additional file 1: Table S4, Subset-2). The average proportion of the methylated miR-137 was also significantly higher in GCs than SMs (Mean ± SD, 26 ± 2 versus 38 ± 2, Paired t-test, P < 0.001). As expected a significant difference in the positive rate or the proportion of methylated miR-9-3, miR-34b, miR-210, and miR-200b was not observed between GC and SM samples. These results confirmed that miR-9-1 and miR-137 methylation was a tumor-specific event and that miR-9-3, miR-34b, and miR-210 methylation, as well as miR-200b demethylation, was a field-effect that occurred during gastric carcinogenesis.
Furthermore, the positive rate of methylated miR-203 and miR-375 gradually increased from normal to gastritis to SM samples, but significantly decreased in GCs compared with SMs (Pearson Chi-square test, miR-203, P = 0.007; miR-375, P = 0.031; Table 1). In the Subset-2 samples, we also analyzed miR-375 methylation and found more miR-375 methylation in SMs than in GCs again (Pearson Chi-square test, P = 0.034; Additional file 1: Table S4). These data imply that miR-375 (and miR-203) methylation is not GC-specific and might be one kind of host adaptation in the non-malignant tissues to the development of GCs.
miR methylation and H. Pyloriinfection
Inversed relationship between miRmethylation of CpG islands and their corresponding expression levels
We further analyzed the miRNA levels of 20 pairs of fresh GC and SM samples and found a significantly higher expression level of miRNA-200b, miRNA-375, and miRNA-210 in SMs than in GCs (Paired t-test: Ps≤0.030; Additional file 1: Figure S12). The miRNA-137 levels in SMs were also higher than those in GCs, but was not statistically significant (P = 0.059). The expression levels of miRNA-9 and miRNA-34b were similar between SMs and GCs. Most importantly, an inverse relationship between miR methylation and the corresponding expression level was observed for miR-9-1, miR-9-3, miR-137, and miR-200b in these gastric tissue samples (Spearman’s Rank Correlation analysis, miR-9-1, r s = −0.533, P = 0.001; miR-9-3, r s = −0.464, P = 0.004; miR-137, r s = −0.378, P = 0.019; miR-200b, r s = −0.409, P = 0.010; Figure 3H-K). A weak inverse methylation-expression relationship was also found for miR-375 in these tissue samples (r s =0.287, P = 0.085; Figure 3O). Such a relationship was not observed for miR-34b and miR-210 (Figure 3L, N).
miRmethylation correlated to clinicopathological characteristics of GC patients
Comparison of methylation of 7 miR CpG islands in 112 GC samples from patients with various clinicopathological characteristics
Positive rate (%)
Proportion, Median* [25-75%]
Positive rate (%)
Positive rate (%)
Positive rate (%)
Positive rate (%)
Positive rate (%)
Positive rate (%)
≤60 (n = 49)
34 [16–48] b
>60 (n = 63)
Male (n = 80)
Female (n = 32)
No (n = 100)
Yes (n = 9)
Cardiac (n = 34)
43 [35–51] b
49 [28–61] b
Non-cardiac (n = 78)
Well/Mod. (n = 29)
Poor (n = 78)
No (n = 60)
10 [3–19] c
Yes (n = 49)
I-II (n = 40)
10 [3–28] c
III-IV (n = 59)
Depth of invasion
T1-2 (n = 25)
T3 (n = 53)
T4 (n = 25)
Lymph node metastasis
N0 (n = 57)
53 [43–57] c
N1-3 (n = 51)
M0 (n = 77)
9 [5–17] c
M1 (n = 31)
The expression patterns exhibited by the miR genes reflect cell lineages and differentiation status of tumor tissue . Increasing attention is currently being focused on miRNAs due to their contribution to maintaining the pluripotency of cancer stem/initiating cells and their application potential as molecular therapy targets and biomarkers. However, the regulatory mechanisms of miR expression in cells and alterations of miRNA levels in tumors are far from clear. In the present study, we characterized the methylation status of 9 representative miR CpG islands in human gastric tissues with various pathological changes and found that abnormal methylation of 5 miR CpG islands (miR-9-1, miR-9-3, miR-34b/c, miR-137, and miR-210) and demethylation of miR-200b CpG island correlated with the development of GC. Furthermore, methylation of the miR-203 and miR-375 CpG islands might be one kind of host adaptations to gastric carcinogenesis. Most importantly, we found that methylation of all these CpG islands was inversely correlated with the expression of these miR genes in a panel of cultured cell lines. To the best of our knowledge, this is the first comprehensive study to illustrate the inverse relationship between the methylation of miR CpG islands and their expression.
Transcription of protein-coding genes is mainly regulated by transcription factors and the accessibility of their binding sites in the promoter regions. It is well known that methylation of CpG islands around the TSS can block transcription factor binding by decreasing the accessibility of these sites, thus inactivate gene expression epigenetically. Bioinformatic analysis shows that 129 of 721 (18%) of the human miR genes in the miRBase (Release 14.0) are CpG island-related (Additional file 1: Table S1). Of the CpG related miR genes, 50 locate within CpG islands and 9 closely flank CpG islands with a <600-bp CpG-sporadic interval; an additional 70 miR genes are near CpG islands with a 0.6 ~ 10-kb CpG-sporadic interval. It has been suggested that methylation of some miR CpG islands might be inversely correlated to their expression [12–17]. However, a solid relationship between miR methylation and expression has not been thoroughly established as only weak supporting evidence has been provided in many of the previous studies, as we have summarized for 9 tested miR genes/clusters (extragenic miR-9-3, miR-137, miR-200b/200a/429, miR-203, miR-375; intragenic miR-9-1, miR-34b/c, miR-193b/365-1, and miR-210) in this present study (Additional file 1: Table S2) [19–27]. One frequently used evidence is that methylated miR genes could be reactivated by inhibitors of DNA methyltransferase (5-aza-cytosine or 5-aza-deoxycytosine) because these cytosine analogues, which cannot be methylated in DNA, inhibit maintenance methylation through direct replacement of methylation targets (cytosine residues). However, this method is not adequate as methyltransferases inhibition that may indirectly reactivate transcription of both CpG island-free genes and methylated genes through DNA damage and repair pathways .
To investigate systemically if methylation of miR CpG islands represses transcription of miR genes, various cell lines and tissue samples with different methylation status of representative miR CpG islands must be used. Thus, instead of randomly selecting candidates from the 129 miR CpG islands, we first screened a set of CpG islands of miR genes that may be abnormally methylated during gastric carcinogenesis. Initially the methylation status of 9 miR CpG islands in human gastric tissues with various pathological changes was analyzed with DHPLC. As is consistent with others’ reports [14, 15, 19, 25, 27, 36–40], miR-9-1, miR-9-3, miR-34b, miR-137, and miR-375 methylation was observed in gastric carcinogenesis in the present study. We also observed methylation changes in miR-200b, miR-193b, miR-203, and miR-210 CpG islands in the development of GCs that has not been previously reported. Interestingly, we found that the proportion of demethylated miR-200b gradually increased significantly in gastric tissues along with the severity of these changes. This suggests that miR-200b demethylation (or hypomethylation) may be involved in gastric carcinogenesis. In addition, miR-203 and miR-375 methylation increased gradually in gastritis and SM samples, but decreased in GC samples. This implies that miR-203 and miR-375 methylation is not a GC-specific event, but rather a host adaptation to gastric carcinogenesis.
Expression of some intragenic miR genes is coordinately regulated by the transcriptional mechanism of their host genes. However, a certain proportion of intragenic miR genes has their own TSS and transcribe in a host gene-independent pattern [9, 41–43]. Unfortunately, the TSS sites have not been characterized for most extragenic/intergenic miR genes. It is reported that expression levels of some miR genes (including intragenic miR-152 and miR-34a/b/c and extragenic miR-203, miR-124-1/124-2, miR-129-2, and miR-181c) inversely correlate with methylation of their corresponding CpG islands [11, 13, 14, 16, 27, 38, 44]. In the present study, we found that methylation or demethylation of all 7 tested miR CpG islands (GC-related miR-9-1, miR-34b, miR-9-3, miR-137, miR-210, miR-200b and host-related miR-375) was consistently, inversely correlated to a statistically significant level with their corresponding miRNA levels in a number of human cell lines in vitro. Such an inverse relationships could also be observed for the miR-9-1, miR-9-3, miR-137, and miR-200b CpG islands in gastric tissue samples in vivo. Although expression of intragenic miR-9-1 is host gene-independent , we cannot exclude the possibility that the expression of miR-34b and miR-210 genes may also be coordinately controlled by regulatory mechanisms of their host genes. An unbiased correlation study using a customized, oligo microarray to detect methylation of all miR CpG islands and expression of intragenic miR genes and their host genes in various cell lineages at different differentiation stages may be useful to clarify which miR genes may be expressed in a host-gene dependent way. Taken together, these data strongly suggest that the methylation status of miR CpG islands could play a crucial role in the regulation of the expression of the related miR genes.
Generally, transcriptional inactivation of a miR gene by methylation in a few cells should not lead to a visible decrease in the miRNA level in a tissue sample because the majority of cells will still exhibit normal miRNA expression. This help explain why a significant inverse methylation-expression relationship was not observed for miRNA-34b, miRNA-210, or miRNA-375 as the average proportion of methylated miR-34b, miR-210, or miR-375 in SM and GC samples was relatively low (2% ~ 15%). Hypermethylation of genes in some cell populations and the concomitant over-expression of these genes in other cell populations are often reported in the same tissue samples with chronic inflammation . Therefore, both the prevalence of miR methylation and the total mature miRNA levels may be useful predictors for miR inactivation and expression in tissue samples, especially in the case of low proportion of methylated miR. Although other factors certainly affect the miRNA expression levels in cells, the inverse relationship consistently observed in this present study between miR methylation and mature miRNA expression level in a number of monoclonal cell lines suggests that the cellular heterozygosity may account for the inconsistent methylation-expression relationship of some miR in tissue samples.
The miR-9-1, miR-9-2, and miR-9-3 genes all encode the same mature miRNA-9 that affects cell migration and proliferation in a tumor type-specific pattern through the NF-κB1 Snail E-cadherin [45, 46]. Abnormal miR-9-1 and miR-9-3 methylation is frequently reported in many cancers including GCs [15, 19, 40]. In the present study, we found that the positive rate of miR-9-1 and miR-9-3 methylation for all 112 GCs was significantly higher than that in 50 non-malignant tissues collected from 37 gastritis patients and 13 healthy controls. In all the tested samples, the prevalence of miR-9-3 methylation is significantly higher than that of miR-9-1. It has been reported that miR-9-3 methylation correlates with poor clinical outcomes for GC patients . However, our study failed to make the same correlation. In contrast, a significant higher of the positive rate and proportion of methylated miR-9-1, but not methylated miR-9-3, was observed in GCs than SMs. This data indicates that miR-9-1 methylation may be a late cancer-specific event, while miR-9-3 methylation may be an early field effect during gastric carcinogenesis. Using miR-9-1 methylation as a biomarker for GC detection, we found a sensitivity and specificity 62% (69 of 112) and 96% (46 of 48) could be achieved, respectively. Because the positive rate of miR-9-1 methylation in GCs or SMs from stage I ~ II GC patients was similar to that of stage III ~ IV GC patients (GCs: 70% versus 59%; SMs: 40% versus 25%), it would be clinically beneficial to determine if miR-9-1 methylation in gastric juice or peripheral blood plasma could be used as a biomarker for prediction of malignant transformation of precancerous lesions of the stomach and early diagnosis of GCs. Although miR-9-1 methylation does not significantly correlate with differentiation, local invasion, metastasis, and pTNM stages in the screening cohort, it does significantly correlate with a longer overall survival of GC patients in univariate and multivariate analysis. Combination with the methylation status of other miR genes failed to strengthen the prediction power of miR-9-1 methylation on the survival of these patients. Therefore, miR-9-1 methylation might be an independent predictor of survival.
miR-137 has been reported to target the CtBP1, a co-repressor of various tumor suppressor genes . Many studies have reported miR-137 methylation in head and neck and colorectal carcinomas [22, 23, 48, 49]. We found that miR-137 methylation was also very common among gastritis lesion, SM, and GC samples (63% ~ 96%), and the positive rate of miR-137 methylation increased gradually in gastric tissues along with the severity of pathological changes. Therefore, miR-137 methylation may be another early epigenetic event that occurs during gastric carcinogenesis. Because the proportion of methylated miR-137 showed a positive correlation with the invasiveness of GCs, miR-137 methylation may also affect the progression of the tumor. When compared to the GC patients without miR-137 methylation, GC patients with miR-137 methylation were more likely to have a poor overall survival; however, this effect was not statistically significant. This is consistent previous study that has reported that miR-137 methylation (by MSP) correlated to the overall survival of 67 patients with head and neck carcinomas .
miR-34b and miR-34c constitute a miR gene cluster. Previous studies have suggested that mature miRNA-34b and miRNA-34c are targets of P53 . As a tumor suppressor gene, miR-34b/c methylation has also frequently been reported in many carcinomas including GCs [14, 51, 52]. miR-34b methylation has been reported to relate to the invasiveness of non-small lung carcinoma, colorectal carcinoma, GC, and melanoma [53–55]. In the present study, we did not find a significant correlation between miR-34b methylation and GC invasion; however, a higher proportion of methylated miR-34b was observed in stage I ~ II GCs than in stage III ~ IV GCs (P = 0.025). The overall survival of GC patients with methylated miR-34b (the proportion > 4%) was likely to be longer than in those patients without the methylation. In addition, we found that the prevalence of miR-34b methylation between SM and GC samples was similar; this is consistent with a recent report that miR-34b methylation might be an early field-effect in the development of GCs .
The miR-200b gene is located in the miR-200b/miR-200a/miR-429 cluster. Its function is related to the processes of epithelium-mesenchymal transition (EMT) by targeting ZEB1 and ZEB2 and results in E-cadherin silencing [56–58]. There is a CpG island upstream of this cluster which contains a 4-kb CpG-sporadic interval. It has been reported that miR-200a/b is hypomethylated and over-expressed in pancreatic cancers and that circulating miRNA-200 may be used as a cancer biomarker. The positive relationship between miR-200b demethylation and over-expression has also been previously found . In the present study, we observed that full demethylation of the miR-200b CpG island was a common field event in both GC and SM samples. Because the proportion of demethylated miR-200b in gastritis/normal biopsies was significantly lower than that in SM and GC samples, miR-200b demethylation might be another potential GC biomarker.
miR-210 is a hypoxia inducible gene which may inhibit cancer cell survival and proliferation through targeting FGFRL1 . It has been reported that miR-210 may directly bind to vacuole membrane protein 1 (VMP1) and promote cancer metastasis . miR-210 methylation has been previously reported to be presented in glioma . We found that miR-210 methylation not only correlated with the severity of gastric pathogenesis, but also correlated with H. pylori infection. According to the possible cause effect relationship between H. pylori infection and the development of GCs, miR-210 inactivation by DNA methylation may play a role in gastric carcinogenesis.
miR-375 promotes cancer cell proliferation through RASD1-ERα pathway . methylation has been previously reported in melonoma , breast cancer , gastric carcinoma  and hepatocellular carcinoma . However, our present study found that for the 106 patients studied, there was a significant higher positive rate and proportion of methylated miR-375 present in SMs compared to GCs. Because significant correlation between miR-375 methylation and H. pylori infection was not observed among the tested gastric samples, we suggest that miR-375 methylation might be a unique host adaptation to the development of GCs.
Previous studies have shown that miR-203 methylation drives H. pylori-associated gastric lymphomagenesis . miR-203 and miR-193b methylation has also been reported in hepatomas and prostate cancer, respectively [12, 37]. We observed that the positive rate of miR-203 and miR-193b methylation increased in normal, gastritis, and SM tissues, but decreased in GCs as did miR-375 methylation pattern. However, correlation between H. pylori infection and miR-203 and miR-193b methylation was not found in any of the gastric tissue samples that were studied. Therefore, we hypothesize that miR-203 and miR-193b methylation may also be a host adaptation to the development of gastritis or GCs.
In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 and miR-137 methylation may be a GC-specific event. Methylation of miR CpG islands may significantly down-regulate their transcription regularly.
We thank Dr. Huidong Shi and Mr. James Wilson at Georgia Health Science University for English language editing. This work is supported by Natural Science Foundation of China (A3 Foresight Program No. 30921140311), National High Technology R&D Program 2006AA02A402, and National Basic Research Program of China (973 Program 2010CB529300 and 2011CB504201).
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