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Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE
Yong Chan Lee |
Professor of Medicine Director, Gastrointestinal Research Laboratory Veterans Affairs Medical Center, Univ. California San Francisco San Francisco, USA |
Jong Pil Im | Seoul National University College of Medicine, Seoul, Korea |
Robert S. Bresalier | University of Texas M. D. Anderson Cancer Center, Houston, USA |
Steven H. Itzkowitz | Mount Sinai Medical Center, NY, USA |
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Ji Min Choi1 , Sang Gyun Kim2
, Hyo-Joon Yang3
, Joo Hyun Lim1
, Nam-Yun Cho4
, Woo Ho Kim5
, Joo Sung Kim1,2
, Hyun Chae Jung2
Correspondence to: Sang Gyun Kim
Division of Gastroenterology, Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 03080, Korea
Tel: +82-2-740-8112, Fax: +82-2-743-6701, E-mail: harley1333@hanmail.net
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gut Liver 2020;14(5):571-580. https://doi.org/10.5009/gnl19299
Published online January 3, 2020, Published date September 15, 2020
Copyright © Gut and Liver.
Background/Aims: Epigenetic change is one of the mechanisms that regulates the expression of microRNAs (miRNAs) and is known to play a role in Helicobacter pylori-associated gastric carcinogenesis. We aimed to evaluate the epigenetic changes of miR-200a/b in H. pylori-associated gastric carcinogenesis and restoration after eradication. Methods: The expression and methylation levels of miR-200a/b were evaluated in gastric cancer (GC) cell lines, human gastric mucosa of H. pylori-negative and -positive controls, and H. pyloripositive GC patients. Next, the changes in the expression and methylation levels of miR-200a/b were compared between H. pylori -eradication and H. pylori -persistence groups at 6 months. Real-time reverse transcription-polymerase chain reaction was conducted to investigate the miRNA expression levels, and MethyLight was performed to assess the methylation levels. Results: In the GC cell lines, the level of miR- 200a/b methylation decreased and the level of expression increased after demethylation. In the human gastric mucosa, the miR-200a/b methylation levels increased in the following group order: H. pylori-negative control group, H. pylori-positive control group, and H. pylori-positive GC group. Conversely, the miR-200a/b expression levels decreased in the same order. In the H. pylori -persistence group, no significant changes were observed in the methylation and expression levels of miR-200a/b after 6 months, whereas the level of methylation decreased and the level of expression of miR-200a/b increased significantly 6 months in the H. pylori-eradication group. Conclusions: Epigenetic alterations of miR-200a/b may be implicated in H. pylori -induced gastric carcinogenesis. This field defect for cancerization is suggested to be improved by H. pylori eradication.
Keywords: Helicobacter pylori, MicroRNAs, Methylation, Epigenetic alteration, Stomach neoplasm
Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of global cancer mortality.1 Although the incidence is declining globally, East Asia, including Korea and Japan, remains a region of high incidence of GC.2 It has been widely accepted that GC, especially intestinal-type GC, develops through progressive changes from chronic gastritis to gastric atrophy, intestinal metaplasia, dysplasia, and invasive carcinoma.
Recently, epigenetic changes have been attracting attention as one of the mechanisms of gastric carcinogenesis. One of the most consistent epigenetic changes in human cancer is aberrant DNA methylation, which has also been linked to gastric carcinogenesis.4 Importantly, previous reports have shown that methylation alterations of multiple genes occurs in both
The
Here, we examined whether epigenetic fields related to
Three GC cell lines, AGS, MKN-1, and MKN-45 were obtained from the Korean Cell Line Bank (Seoul, Korea) (Supplementary Table 1). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum, L-glutamine (300 mg/L), 25 mM HEPES, and 25 mM NaHCO3, and plated on day 0. On day 1, cells were treated with 2 μM 5-Aza-2’-deoxycytidine (Sigma-Aldrich and Merck KGaA, Darmstadt, Germany), demethylating agent, and replenished daily with the demethylating agent and medium. Cells were harvested on day 4.
We included 40 patients with
miRNAs were isolated from cell lines and gastric tissues preserved at –80°C using mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized from reverse transcription of the miRNAs using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Quantitative reverse transcription-polymerase chain reaction was carried out using 2 μL of complementary DNA in a total mix of 20 μL containing 10 μL of TaqMan Universal Master Mix II (Applied Biosystems) and analyzed on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Human GAPDH gene served as an internal control and the relative expression levels of miRNAs were calculated using the comparative 2–ΔΔCt method.26 All samples were tested in triplicate.
DNA was isolated from the cell lines and tissues using the LaboPassTM Blood Mini kit (Cosmogenetech, Seoul, Korea) following the manufacturer’s instructions. Bisulfite conversion was performed on 1 μg of genomic DNA using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA), which convert unmethylated cytosine to uracil.
Methylation status of the bisulfite-modified miRNA promoters was analyzed using real-time PCR-based MethyLight assay.27,28 Pairs of primers and probes were designed by the software, Beacon Designer (PREMIER Biosoft International, Palo Alto, CA, USA) (Supplementary Table 2). In both
The Kruskal-Wallis test was recruited for the overall comparison of continuous variables of the three groups (
We measured the levels of miRNA expression and DNA methylation in the GC cell lines, AGS, MKN-1, and MKN-45. To assess the role of methylation in the expression of miRNAs, we examined their expression in three GC cell lines before and after the treatment with the demethylating agent, 5-Aza-2’-deoxycytidine. The levels of
In clinicopathological characteristics, the patients with
We measured the level of methylation and corresponding miRNA expression in the gastric mucosa of the three groups. In the MethyLight assay, the promoter DNA methylation level of
Because of the significant difference in age and sex in the baseline characteristics of each group, further analysis was needed with adjustment of these variables. Promoter methylation and miRNA expression of miRNAs were analyzed using a non-parametric ranked analysis of covariance model with group as a factor, and age and sex as covariates. In this model, the levels of promoter methylation (p=0.001) and expression of
In 40 patients with
In this study, the level of DNA methylation might influence the level of
Recently, epigenetic changes in cancer-related genes, such as tumor suppressor genes, have been proposed to be one of the important processes in
The
The present study revealed that
In this study, the eradication of
We demonstrated that the degree of neutrophil and monocyte infiltration decreased significantly at 6 months after eradication in the
In this study, the methylation levels of
This study has several strengths. To date, this is the first to demonstrate promoter methylation and subsequent dysregulation of
This study also has some limitations. First, a small number of patients were included for analysis. Nevertheless, the differences between the groups were large enough to reach statistical significance. In subgroup analysis of
In conclusion, aberrant DNA methylation of
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03036304); and the SNUH research fund (grant number: 03-2016-0310).
The authors thank Ji Hee Kim (Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea) for technical assistance.
No potential conflict of interest relevant to this article was reported.
Study conception and design, data acquisition/analysis/interpretation, manuscript drafting: J.M.C., S.G.K., N.Y.C. Statistical analysis: J.M.C., H.J.Y. Critical revision of the manuscript: H.J.Y., J.H.L., N.Y.C., W.H.K., J.S.K., H.C.J. Obtained funding, study supervision: S.G.K. All authors read and approved the final manuscript.
Clinicopathological Characteristics of the Subjects
Variable | GCs (n=40) |
controls (n=20) |
p-value* | controls (n=20) |
p-value† |
---|---|---|---|---|---|
Age, yr | 66.5 (59.3–72.0) | 57.0 (48.0–61.0) | <0.001 | 56.5 (47.3–61.8) | <0.001 |
Male sex | 26 (65.0) | 4 (20.0) | 0.002 | 7 (35.0) | 0.028 |
Mucosal atrophy | 0.077 | 0.019 | |||
Absent to mild | 24 (60.0) | 17 (85.0) | 18 (90.0) | ||
Moderate to severe | 16 (40.0) | 3 (15.0) | 2 (10.0) | ||
Intestinal metaplasia | 0.002 | 0.007 | |||
Absent to mild | 17 (42.5) | 17 (85.0) | 16 (80.0) | ||
Moderate to severe | 23 (57.5) | 3 (15.0) | 4 (20.0) | ||
Neutrophil | 1.000 | <0.001 | |||
Absent to mild | 1 (2.5) | 1 (5.0) | 19 (95.0) | ||
Moderate to severe | 39 (97.5) | 19 (95.0) | 1 (5.0) | ||
Monocyte | 0.595 | <0.001 | |||
Absent to mild | 2 (5.0) | 2 (10.0) | 18 (90.0) | ||
Moderate to severe | 38 (95.0) | 18 (90.0) | 2 (10.0) |
Data are presented as the median (interquartile range) or number (%).
*Comparison between the
Differences in the Promoter Methylation and Expression Levels of miRNAs among the Groups after Adjustment for Covariates
Variable | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Promoter methylation | miRNA expression | Promoter methylation | miRNA expression | |||||||||
df | F | p-value | df | F | p-value | df | F | p-value | df | F | p-value | |
Age | 1 | 0.482 | 0.490 | 1 | 0.016 | 0.900 | 1 | 0.004 | 0.952 | 1 | 0.565 | 0.455 |
Sex | 1 | 0.709 | 0.402 | 1 | 1.175 | 0.282 | 1 | 0.329 | 0.568 | 1 | 1.343 | 0.250 |
Group | 2 | 8.104 | 0.001 | 2 | 6.468 | 0.003 | 2 | 8.977 | <0.001 | 2 | 8.415 | 0.001 |
miRNA, microRNA; df, degree of freedom; F, variance ratio.
Clinicopathological Characteristics of the
Characteristic | Baseline | 6-Month follow-up | ||||
---|---|---|---|---|---|---|
(n=20) |
(n=20) |
(n=20) |
(n=20) |
p-value* | ||
Age, yr | 70.0 (56.5–72.8) | 65.0 (60.3–70.8) | - | - | ||
Male sex | 12 (60.0) | 14 (70.0) | - | - | ||
Mucosal atrophy | 0.327 | |||||
Absent to mild | 10 (50.0) | 14 (70.0) | 14 (70.0) | 11 (55.0) | ||
Moderate to severe | 10 (50.0) | 6 (30.0) | 6 (30.0) | 9 (45.0) | ||
Intestinal metaplasia | 0.519 | |||||
Absent to mild | 6 (30.0) | 11 (55.0) | 7 (35.0) | 9 (45.0) | ||
Moderate to severe | 14 (70.0) | 9 (45.0) | 13 (65.0) | 11 (55.0) | ||
Neutrophil | <0.001 | |||||
Absent to mild | 1 (5.0) | 0 | 17 (85.0) | 2 (10.0) | ||
Moderate to severe | 19 (95.0) | 20 (100.0) | 3 (15.0) | 18 (90.0) | ||
Monocyte | 0.001 | |||||
Absent to mild | 1 (5.0) | 1 (5.0) | 9 (45.0) | 0 | ||
Moderate to severe | 19 (95.0) | 19 (95.0) | 11 (55.0) | 20 (100.0) |
Data are presented at the median (interquartile range) or number (%).
*Comparison between the 6-month follow-up results in the
Gut and Liver 2020; 14(5): 571-580
Published online September 15, 2020 https://doi.org/10.5009/gnl19299
Copyright © Gut and Liver.
Ji Min Choi1 , Sang Gyun Kim2
, Hyo-Joon Yang3
, Joo Hyun Lim1
, Nam-Yun Cho4
, Woo Ho Kim5
, Joo Sung Kim1,2
, Hyun Chae Jung2
1Department of Internal Medicine, Healthcare Research Institute, Seoul National University Hospital Healthcare System Gangnam Center, 2Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 3Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, 4Laboratory of Epigenetics, Cancer Research Institute, and 5Department of Pathology, Seoul National University College of Medicine, Seoul, Korea
Correspondence to:Sang Gyun Kim
Division of Gastroenterology, Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 03080, Korea
Tel: +82-2-740-8112, Fax: +82-2-743-6701, E-mail: harley1333@hanmail.net
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background/Aims: Epigenetic change is one of the mechanisms that regulates the expression of microRNAs (miRNAs) and is known to play a role in Helicobacter pylori-associated gastric carcinogenesis. We aimed to evaluate the epigenetic changes of miR-200a/b in H. pylori-associated gastric carcinogenesis and restoration after eradication. Methods: The expression and methylation levels of miR-200a/b were evaluated in gastric cancer (GC) cell lines, human gastric mucosa of H. pylori-negative and -positive controls, and H. pyloripositive GC patients. Next, the changes in the expression and methylation levels of miR-200a/b were compared between H. pylori -eradication and H. pylori -persistence groups at 6 months. Real-time reverse transcription-polymerase chain reaction was conducted to investigate the miRNA expression levels, and MethyLight was performed to assess the methylation levels. Results: In the GC cell lines, the level of miR- 200a/b methylation decreased and the level of expression increased after demethylation. In the human gastric mucosa, the miR-200a/b methylation levels increased in the following group order: H. pylori-negative control group, H. pylori-positive control group, and H. pylori-positive GC group. Conversely, the miR-200a/b expression levels decreased in the same order. In the H. pylori -persistence group, no significant changes were observed in the methylation and expression levels of miR-200a/b after 6 months, whereas the level of methylation decreased and the level of expression of miR-200a/b increased significantly 6 months in the H. pylori-eradication group. Conclusions: Epigenetic alterations of miR-200a/b may be implicated in H. pylori -induced gastric carcinogenesis. This field defect for cancerization is suggested to be improved by H. pylori eradication.
Keywords: Helicobacter pylori, MicroRNAs, Methylation, Epigenetic alteration, Stomach neoplasm
Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of global cancer mortality.1 Although the incidence is declining globally, East Asia, including Korea and Japan, remains a region of high incidence of GC.2 It has been widely accepted that GC, especially intestinal-type GC, develops through progressive changes from chronic gastritis to gastric atrophy, intestinal metaplasia, dysplasia, and invasive carcinoma.
Recently, epigenetic changes have been attracting attention as one of the mechanisms of gastric carcinogenesis. One of the most consistent epigenetic changes in human cancer is aberrant DNA methylation, which has also been linked to gastric carcinogenesis.4 Importantly, previous reports have shown that methylation alterations of multiple genes occurs in both
The
Here, we examined whether epigenetic fields related to
Three GC cell lines, AGS, MKN-1, and MKN-45 were obtained from the Korean Cell Line Bank (Seoul, Korea) (Supplementary Table 1). Cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum, L-glutamine (300 mg/L), 25 mM HEPES, and 25 mM NaHCO3, and plated on day 0. On day 1, cells were treated with 2 μM 5-Aza-2’-deoxycytidine (Sigma-Aldrich and Merck KGaA, Darmstadt, Germany), demethylating agent, and replenished daily with the demethylating agent and medium. Cells were harvested on day 4.
We included 40 patients with
miRNAs were isolated from cell lines and gastric tissues preserved at –80°C using mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized from reverse transcription of the miRNAs using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Quantitative reverse transcription-polymerase chain reaction was carried out using 2 μL of complementary DNA in a total mix of 20 μL containing 10 μL of TaqMan Universal Master Mix II (Applied Biosystems) and analyzed on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Human GAPDH gene served as an internal control and the relative expression levels of miRNAs were calculated using the comparative 2–ΔΔCt method.26 All samples were tested in triplicate.
DNA was isolated from the cell lines and tissues using the LaboPassTM Blood Mini kit (Cosmogenetech, Seoul, Korea) following the manufacturer’s instructions. Bisulfite conversion was performed on 1 μg of genomic DNA using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA), which convert unmethylated cytosine to uracil.
Methylation status of the bisulfite-modified miRNA promoters was analyzed using real-time PCR-based MethyLight assay.27,28 Pairs of primers and probes were designed by the software, Beacon Designer (PREMIER Biosoft International, Palo Alto, CA, USA) (Supplementary Table 2). In both
The Kruskal-Wallis test was recruited for the overall comparison of continuous variables of the three groups (
We measured the levels of miRNA expression and DNA methylation in the GC cell lines, AGS, MKN-1, and MKN-45. To assess the role of methylation in the expression of miRNAs, we examined their expression in three GC cell lines before and after the treatment with the demethylating agent, 5-Aza-2’-deoxycytidine. The levels of
In clinicopathological characteristics, the patients with
We measured the level of methylation and corresponding miRNA expression in the gastric mucosa of the three groups. In the MethyLight assay, the promoter DNA methylation level of
Because of the significant difference in age and sex in the baseline characteristics of each group, further analysis was needed with adjustment of these variables. Promoter methylation and miRNA expression of miRNAs were analyzed using a non-parametric ranked analysis of covariance model with group as a factor, and age and sex as covariates. In this model, the levels of promoter methylation (p=0.001) and expression of
In 40 patients with
In this study, the level of DNA methylation might influence the level of
Recently, epigenetic changes in cancer-related genes, such as tumor suppressor genes, have been proposed to be one of the important processes in
The
The present study revealed that
In this study, the eradication of
We demonstrated that the degree of neutrophil and monocyte infiltration decreased significantly at 6 months after eradication in the
In this study, the methylation levels of
This study has several strengths. To date, this is the first to demonstrate promoter methylation and subsequent dysregulation of
This study also has some limitations. First, a small number of patients were included for analysis. Nevertheless, the differences between the groups were large enough to reach statistical significance. In subgroup analysis of
In conclusion, aberrant DNA methylation of
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03036304); and the SNUH research fund (grant number: 03-2016-0310).
The authors thank Ji Hee Kim (Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea) for technical assistance.
No potential conflict of interest relevant to this article was reported.
Study conception and design, data acquisition/analysis/interpretation, manuscript drafting: J.M.C., S.G.K., N.Y.C. Statistical analysis: J.M.C., H.J.Y. Critical revision of the manuscript: H.J.Y., J.H.L., N.Y.C., W.H.K., J.S.K., H.C.J. Obtained funding, study supervision: S.G.K. All authors read and approved the final manuscript.
Table 1. Clinicopathological Characteristics of the Subjects
Variable | p-value* | p-value† | |||
---|---|---|---|---|---|
Age, yr | 66.5 (59.3–72.0) | 57.0 (48.0–61.0) | <0.001 | 56.5 (47.3–61.8) | <0.001 |
Male sex | 26 (65.0) | 4 (20.0) | 0.002 | 7 (35.0) | 0.028 |
Mucosal atrophy | 0.077 | 0.019 | |||
Absent to mild | 24 (60.0) | 17 (85.0) | 18 (90.0) | ||
Moderate to severe | 16 (40.0) | 3 (15.0) | 2 (10.0) | ||
Intestinal metaplasia | 0.002 | 0.007 | |||
Absent to mild | 17 (42.5) | 17 (85.0) | 16 (80.0) | ||
Moderate to severe | 23 (57.5) | 3 (15.0) | 4 (20.0) | ||
Neutrophil | 1.000 | <0.001 | |||
Absent to mild | 1 (2.5) | 1 (5.0) | 19 (95.0) | ||
Moderate to severe | 39 (97.5) | 19 (95.0) | 1 (5.0) | ||
Monocyte | 0.595 | <0.001 | |||
Absent to mild | 2 (5.0) | 2 (10.0) | 18 (90.0) | ||
Moderate to severe | 38 (95.0) | 18 (90.0) | 2 (10.0) |
Data are presented as the median (interquartile range) or number (%).
*Comparison between the
Table 2. Differences in the Promoter Methylation and Expression Levels of miRNAs among the Groups after Adjustment for Covariates
Variable | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Promoter methylation | miRNA expression | Promoter methylation | miRNA expression | |||||||||
df | F | p-value | df | F | p-value | df | F | p-value | df | F | p-value | |
Age | 1 | 0.482 | 0.490 | 1 | 0.016 | 0.900 | 1 | 0.004 | 0.952 | 1 | 0.565 | 0.455 |
Sex | 1 | 0.709 | 0.402 | 1 | 1.175 | 0.282 | 1 | 0.329 | 0.568 | 1 | 1.343 | 0.250 |
Group | 2 | 8.104 | 0.001 | 2 | 6.468 | 0.003 | 2 | 8.977 | <0.001 | 2 | 8.415 | 0.001 |
miRNA, microRNA; df, degree of freedom; F, variance ratio.
Table 3. Clinicopathological Characteristics of the
Characteristic | Baseline | 6-Month follow-up | ||||
---|---|---|---|---|---|---|
p-value* | ||||||
Age, yr | 70.0 (56.5–72.8) | 65.0 (60.3–70.8) | - | - | ||
Male sex | 12 (60.0) | 14 (70.0) | - | - | ||
Mucosal atrophy | 0.327 | |||||
Absent to mild | 10 (50.0) | 14 (70.0) | 14 (70.0) | 11 (55.0) | ||
Moderate to severe | 10 (50.0) | 6 (30.0) | 6 (30.0) | 9 (45.0) | ||
Intestinal metaplasia | 0.519 | |||||
Absent to mild | 6 (30.0) | 11 (55.0) | 7 (35.0) | 9 (45.0) | ||
Moderate to severe | 14 (70.0) | 9 (45.0) | 13 (65.0) | 11 (55.0) | ||
Neutrophil | <0.001 | |||||
Absent to mild | 1 (5.0) | 0 | 17 (85.0) | 2 (10.0) | ||
Moderate to severe | 19 (95.0) | 20 (100.0) | 3 (15.0) | 18 (90.0) | ||
Monocyte | 0.001 | |||||
Absent to mild | 1 (5.0) | 1 (5.0) | 9 (45.0) | 0 | ||
Moderate to severe | 19 (95.0) | 19 (95.0) | 11 (55.0) | 20 (100.0) |
Data are presented at the median (interquartile range) or number (%).
*Comparison between the 6-month follow-up results in the