Increased 5-hydroxymethylcytosine is a favorable prognostic factor of Helicobacter pylori-negative gastric cancer patients

INTRODUCTION

Gastric cancer (GC) is a serious disease with over 1 million estimated new cases annually around the world, and it is the fifth most diagnosed malignancy worldwide[1]. Due to the symptoms in early stage being obscure, most GC patients are diagnosed at middle or late stage, which causes higher mortality rates, and accounted for 769000 deaths globally in 2020[1].

Recent comprehensive analyses showed that many GC-related pathways are more frequently altered by aberrant DNA methylation than by mutations[2], and the degree of accumulation of aberrant DNA methylation is highly correlated with GC risk[3,4].

(

) was identified as a class I carcinogen leading to gastric adenocarcinoma by the World Health Organization[5].

-induced chronic inflammation plays a direct role in the induction of aberrant DNA methylation. The methylation level in an

-positive group was 2.5-34.1 times higher than a negative group.

eradication leads to a decrease in DNA methylation levels[6,7].

A promising method to reverse the progression of GC is effective demethylation treatment. The passive demethylation agents (5-azacytidine/decitabine), which relies on DNA methyltransferase, are not effective in the treatment of solid tumors and have serious side effects. A newly proposed classical active demethylation process involves oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC)and further downstream products by the ten-eleven translocation (TET) family. The median product, 5-hmC, is considered an activating epigenetic marker, and it plays complex roles in gene regulation of tumorigenesis[8-10]. Significant reductions in 5-hmC levels have been found in hematological malignancies, such as breast cancer, colon cancer, prostate cancer and melanoma. A few small size studies analyzed the association between 5-hmC levels and GC, but the evidence is still lacking[11,12],especially for the

-induced GC.

In the current study, we explored the level of 5-hmC and

infection in a relatively large scale GC patient cohort to assess the association between 5-hmC level and the malignant progression of the tumor and the overall survival of GC patients with different

infection status.

MATERIALS AND METHODS

Ethics statement

This study was approved by the Institutional Review Board of the First Hospital of Jilin University. All participants provided written informed consent prior to joining the study.

Study population

He knocked at the door, which was opened by a little old woman who asked, What do you want at this late hour in the midst of this great forest? He said, Haven t you seen a stag about here? Yes, said she, I know the stag well, and as she spoke24 a little dog ran out of the house and began barking and snapping at the stranger

Follow-up

Follow-up for all patients was implemented at 3 mo, 6 mo, 12 mo and annually afterwards until death or the end of the follow-up. Information on general status and postoperative chemotherapy were collected during each follow-up. If the patients had died, the date of death and potential cause were recorded.The duration from the date of surgery to the date of death or the last successful interview date was defined as the survival time. If the patient was lost to follow-up, survival time was defined as the duration from the date of surgery to the date of the last successful interview.

5-hmC quantification and test for H. pylori infection

The genomic DNA from primary tumors and paired noncancerous mucosa tissues were extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The 5-hmC content of genomic DNA was determined with a Quest 5-hmC DNA enzyme-linked immunosorbent assay (ELISA) Kit (Zymo Research, Irvine, CA, United States) according to the manufacturer’s instructions. Assays were performed using 4 μg/mL anti-5-hmC polyclonal antibodies,loading 200 ng of DNA per well. Absorbance at 405 nm was evaluated using a SynergyH1 microplate reader and Gen5 software (BioTek, Winooski, VT, United States). The amount of 5-hmC was calculated as a percentage based on a standard curve generated using kit controls and the median value was used as the cutoff of 5-hmC level category. Values above the median value were considered to be the 5-hmC high group (≥ 0.106%), and those below the median value were considered the 5-hmC low group (<0.106%).

The 5-hmC level was slightly decreased in the

-positive subset compared to the

negative group in our study. It is hypothesized that

infection affects

expression in normal gastric epithelial cells and reduces the genome hydroxymethylation level[19]. Interestingly, higher global 5-hmC levels were associated with GC progression in the

-positive subset. A similar phenomenon was reported that the 5-hmC level in

prostate cancer patients was lower than

+patients, but a higher 5hmC level was associated with tumor progression in

- prostate cancer patients[20]. This could be explained by cells responding to hypoxia inducing a transcriptional program regulated by the

family. Hypoxia together with reactive oxygen species increase global 5-hmC levels by transcriptional activation of

[21,22].

infection induced the expression of hypoxiainducible factor[23], which is required for hypoxic induction of

and global increase of 5-hmC. The proliferation rate of

under aerobic conditions was 3-fold higher than under microaerophilic conditions, and the bacterial growth was more dependent on carbon dioxide than on oxygen[24].

Among the 38 paired tissues, the 5-hmC levels were significantly reduced in tumor tissues (0.076 ±0.048) compared with the matched control tissues (0.110 ± 0.057,

= 0.001) (Figure 2A).

Statistical analysis

We are grateful to all sample donors and the research assistants who helped with sample collection.

Survival curves within each stratification of variables were plotted by the Kaplan-Meier method and compared by log-rank test. The forward stepwise multivariate Cox proportional hazard model was used to evaluate the prognostic role of clinical characteristics and 5-hmC level. Hazard ratios (HRs) with their 95% confidence intervals (Cis) were calculated. All analyses were conducted with the SPSS program(version 21.0; IBM Corp., Armonk, NY, United States) or GraphPad Prism 5.0 (La Jolla, CA, United States). A two-tailed

value < 0.05 indicated statistical significance.

RESULTS

In the present study, 144 GC patients were involved for the final prognostic analysis and were followed up until August 2021. The median survival time was 73.59 mo. During the follow-up period, 75 (52.1%)patients died, 68 (47.2%) patients remained alive, and 1 (0.7%) patient was lost to follow-up (Figure 1).

A commercial ELISA kit for

immunoglobulin G (Biohit, Helsinki, Finland) was used to detect the serum

immunoglobulin G antibodies. The antibody titers were quantified by optical density readings according to the manufacturer’s protocol, and titers higher than the threshold value of 30 EIU were considered as positive for

.

infection.

Among the 144 subjects, there were 99 (68.7%) males, and the median age was 62.82 (range 39–90)years old. The mean 5-hmC level of the 144 GC patients was 0.104 ± 0.062. We investigated possible correlations between 5-hmC levels and general demographic characteristics/routine clinicopathological parameters in the GC patients. The TNM stages (

= 0.012), neural invasion (

= 0.008), age (

= 0.008)and

infection (

= 0.049) were associated with 5-hmC level. Details were shown in Table 1.

Bingo was not intimidated. The impersonal（） nature of the goliath（） now emerging from his own home - attacking and repelling him - only added further fuel to the fury burning in his brain. He grabbed the mop head in his jaws, and began a fiery, frenzied show of temper and torment as he mauled and shook it. My Father was forced eventually to concede that the armoured assault only gained a little ground in what was a war of attrition. The yard was narrow by the kitchen, and Bingo could not get past the steadily advancing pushchair, but as the end of the house was reached, the yard opened up, and Bingo could attack from the sides.

For the 144 GC patients, the results of

infection examination showed that 89 (61.8%) subjects were positive and 55 (38.2%) subjects were negative. We compared the 5-hmC level between

positive infection and

-negative infection groups. It showed that the 5-hmC level was reduced in the

-positive group, but the

value was at the boundary of significance (

= 0.067, Figure 2B).

We further investigated the association between 5-hmC level and characteristics stratified by

infection status. We found that 5-hmC levels were higher in patients aged more than 60 years (

=0.009), with neural invasion positive (

= 0.002), with low histological grade (

= 0.042) or with later TNM stage (

= 0.012) in the

-positive subset, but no significant associations were observed in the

-negative subset except sex (Table 2).

14. Slipped from the palace: The princess in this story is by no means passive such as many of her better known fairy tale counterparts. She attempts to protect herself as much as she can from the threat of immorality79. When she fails, she flees the palace and the threat, abandoning her birthright and the luxury of her life.

Overall survival analyses were performed in total patients and patient stratification by

infection status or TNM stage. The results of the Kaplan-Meier analysis showed that the 5-hmC level was not associated with overall survival in total patients or

-negative or positive groups (log rank

values were 0.406, 0.094 and 0.763, respectively, Figure 3A-C). Furthermore, the 5hmC high level was associated with longer overall survival time compared with the 5hmC low group in the TNM stageⅠ and Ⅱ subgroup, and log rank test showed the survival curves were significantly different (log rank

= 0.037, Figure 3D), but the association was not significant in the TNM stage Ⅲ subgroup (log rank

=0.547, Figure 3E).

A total of 158 patients with histologically diagnosed GC who underwent radical gastrectomy at the Department of Gastric and Colorectal Surgery in the First Hospital of Jilin University (Changchun,China) during 2007 to 2017 were recruited in this cohort study. For each patient, 5 mL of peripheral blood before surgery and 0.5 cm

of tumor tissue were collected. Among the patients, 38 specimens of 0.5 cm

adjacent tissue were collected during the operation. All patients did not undergo chemotherapy or radiotherapy before surgery. Demographic information (sex, age) and principal clinical pathological information (histological grade, TNM stage, tumor size, neural invasion, vascular invasion,

) were collected. The tumor histological grade was evaluated by the World Health Organization criteria. TNM stages were classified according to the 8

edition of the TNM staging system of the Union for International Cancer Control/American Joint Committee on Cancer (2017). Patients with the following conditions were excluded from this study:(1) Patients with distant metastasis or a positive surgical margin; (2) Patients who died due to complications of the surgical procedure during the perioperative period; and (3) Patients who were lost at the first time of interview.

In the full patient set, 5-hmC high level was a significant favorable predictor of overall survival in multivariate Cox regression analysis (HR = 0.61, 95%CI:0.38-0.98,

= 0.040) after adjustment for tumor size, histological grade and TNM stage (Table 3).

Ah, replied the hound,7 as I am old, and daily grow weaker, and no longer can hunt, my master wanted to kill me, so I took to flight; but now how am I to earn my bread?

Multivariate Cox regression analysis for overall survival was also performed in GC patients stratified by

infection status or TNM stage. In the

negative GC subgroup, increased 5-hmC level was a favorable prognostic factor in the multivariate Cox regression analysis (HR = 0.30, 95%CI:0.13-0.68,

= 0.004) (Table 4), which indicated that higher 5-hmC level was an independent significantprotective factor of overall survival time in patients without

infection. However, within the

-positive group, we did not observe any significant association between 5-hmC level and GC patient prognosis.

Among patients with TNM stage I or II, increased 5-hmC level was associated with favorable prognosis after adjustment for sex in the multivariate Cox regression analysis (HR = 0.32, 95%CI:0.13-0.77,

= 0.011). However, no significant association was observed between 5-hmC level and the prognosis in patients with TNM stage Ⅲ (Table 5).

DISCUSSION

Long-time

infection leads to chronic inflammation and further aberrant DNA methylation,which plays an important role in tumorigenesis of GC. The global prevalence of

reported by a meta-analysis across individual countries varied from 18.9% to 87.7%, and the prevalence in China was 55.8% (95%CI:51.8%-59.9%)[13]. Among our 144 GC patients, 89 (61.8%) patients were defined as

-positive by ELISA. The infection rate was slightly higher than the prevalence in the general Chinese population but was similar to the previously reported prevalence in GC patients[14,15],indicating that our study cohort was representative.

DNA methylation/hydroxymethylation is one of the most widely studied epigenetic modifications and has been shown to play significant roles in tumorigenesis and prognosis[16]. Previous studies have shown that aberrant DNA methylation is a common event and a strong candidate mechanism for early nongenetic alterations in GC[17]. Nevertheless, the reports of DNA hydroxymethylation and GC are limited to several small studies.

We estimated the 5-hmC level with an absolutely quantitative method ELISA, which is more objective than the semi-quantitative evaluation system of immunohistochemistry. The results showed that the 5-hmC level was downregulated in GC tissues compared with matched control tissues, which revealed that it was associated with the occurrence of GC and is consistent with previous reports[18]. Although some evidence has emerged about the potential progression and prognostic implications of 5-hmC level in GC[12], very few studies have evaluated the association stratified by

infection status. The present study was performed on a well-characterized cohort to simultaneously evaluate the level of 5-hmC in GC patients and subsets stratified by

infection to assess the association between 5-hmC levels and the susceptibility or prognosis of GC in order to provide more evidence for the effect of

-infection DNA hydroxymethylation on GC.

28. Drunken hussar: A hussar is a European solider, usually a cavalry72 officer. Von Franz points out that the hussar, symbolizes a brutal73 outburst of emotion. The wild ungovernable animus smashes everything, showing clearly that such an exhibition of her unconscious nature does not work (175). This is also another aspect of Wotan (von Franz 175). According to von Franz, the horse the hussar rides is connected to the animus and respects instinctive74 animal nature (176).Return to place in story.

This interesting phenomenon and potential mechanism suggested to us that the 5-hmC level changed due to

infection and was not simply one direction but complicated. Therefore, it was essential to assess the association between 5-hmC and the prognosis of GC patients in negative or positive

infection. Our results first showed that reduced 5-hmC was associated with poor prognosis in all GC patients, which was consistent with previous studies[11,12]. Furthermore, in

-negative GC patients, the 5-hmC level was a significant predictor of prognosis, independent of routine clinicopathological factors. But in contrast, 5-hmC had no prediction value of prognosis in

-positive GC patients. These results highlight the importance of

stratification in GC biomarker studies.Similarly to our results, the study conducted in prostate cancer patients also showed that the prognostic predictor value of 5-hmC was discrepant in

and

prostate cancer patients[20]. Together with our results, it supports potential prognostic implications of 5-hmC as cancer subtype-specific.

In this study, the association between 5-hmC level and the prognosis of GC patients was not significant in the Kaplan-Meier analysis, which could not be adjusted for potential confounders.However, it showed significant association in the multivariate Cox regression after the confounders such as TNM stage were adjusted. This indicated that the clinical characteristics such as TNM stage(which is strongly associated with the prognosis of GC patients) confused the relationship between 5-hmC level and the prognosis. This conclusion was further supported by the Cox regression results of TNM stage stratified analysis.

Several limitations should be mentioned of the present study. First, our study was based at a single center. The prognostic value of 5-hmC in

-negative but not positive GC patients’ needs to be validated in larger and multicenter GC patient cohorts. Another limitation of our study is the lack of data of 5-methylcytosine and enzymes related to 5-hmC regulation for our sample set. Thus, we have not investigated the correlation between them, which should be investigated in future studies.

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CONCLUSION

5 -hmC level was a significant predictor of the prognosis of GC patients without

infection,independent of routine clinicopathological factors.

ARTICLE HIGHLIGHTS

Research background

Most gastric cancer (GC) patients are diagnosed at middle or late stage because the symptoms in early stage are obscure, which causes higher mortality rates of GC. Analyses show that aberrant DNA methylation is highly correlated with GC risk. Helicobacter pylori (H. pylori) was identified as a class I carcinogen leading to gastric adenocarcinoma, and H. pylori-induced chronic inflammation plays a direct role in the induction of aberrant DNA methylation. The median demethylation product 5-hydroxymethylcytosine (5-hmC) is considered as an activating epigenetic marker, and it plays complex roles in gene regulation of tumorigenesis.

Christians58 have the Trinity, the Chinese have the Great Triad (man, heaven, earth), and the Buddhists59 have the Triple Jewel (Buddha, Dharma, Sanga)

Before the fiddler had time to think, he was ushered9 into a little room by invisible hands, and there a table was spread for him with all the delicious food he had seen cooking in the kitchen

Research motivation

A few small studies analyzed the association between 5-hmC levels and GC, but the evidence is lacking,especially for H. pylori-induced GC.

Research objectives

Exploring the association between 5-hmC level and the progression and prognosis of GC patients with or without H. pylori infection.

Research methods

This was a retrospective cohort study to estimate the predicted value of 5-hmC level in the progression and prognosis of GC patients with different H. pylori infection status.

Research results

A high level of 5-hmC was an independent significant favorable predictor of overall survival in the entire GC patient cohort (hazard ratio = 0.61, 95% confidence interval:0.38-0.98,

= 0.040), the

negative GC subgroup (hazard ratio = 0.30, 95% confidence interval:0.13-0.68,

= 0.004) and GC patients with early TNM stage (hazard ratio = 0.32, 95% confidence interval:0.13-0.77,

= 0.011).

Research conclusions

5 -hmC level was a significant predictor of the prognosis of GC patients without

infection.

Research perspectives

“I always sleep with the knife by me,” said the robber-girl. “No one knows what may happen. But now tell me again all about little Kay, and why you went out into the world.”

Continuous variables that followed a normal distribution were shown as the mean ± standard deviation.Independent samples were compared by two-sample

-test, and matched-paired samples were compared by paired

-test. Categorical variables were presented as frequencies with percentages and were compared with the

test or Fisher’s exact test when appropriate.

Cao XY conceived and designed the study; Fu YL and Wu YH conducted data analysis and drafted the manuscript, and they contributed equally to this work; Cao DH, Jia ZF and Shen A collected data and biospecimens on gastric cancer patients; Jiang J measured the level of 5-hmC; All authors approved the final version for submission.

National Natural Science Foundation of China, No. 81874279; Scientific and Technological Development Program of Jilin Province, No. 20190201093JC and No. 20200201326JC; Jilin Province Department of Finance, No. JLSWSRCZX2020-010; and Youth Development Fund from First Hospital of Jilin University, No.JDYY11202021.

This study was approved by the First Hospital of Jilin University Institutional Review Board (Approval No.2021-493).

Patients were not required to give informed consent to the study because the analysis used anonymous clinical data that were obtained after each patient agreed to treatment by written consent.

A large-scale GC patient cohort to assess the association between the level of 5-hmC and the prognosis of GC patients, especially for different

infection status, should be conducted.

All authors declared no competing financial interests.

Technical appendix, statistical code, and dataset available from the corresponding author at email jd3d2ub@jlu.edu.cn.

Joyce Thomas comments on the trickery and imagery of the bone. The bone provides the imagery of deprivation112 and starvation, one of the primary themes of the tale. Also, the bone Hansel uses imitates the fate awaiting his flesh (the bone could well be the gnawed113 remains114 of the cage s previous occupant) (Thomas 1989).Return to place in story.

The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items.

This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BYNC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is noncommercial. See:https://creativecommons.org/Licenses/by-nc/4.0/

It s just a small, white envelope stuck among the branches of our Christmas tree. No name, no identification, no inscription（，）. It has peeked1 through the branches of our tree for the past 10 years or so.

China

Ying-Li Fu 0000-0003-0455-5962; Yan-Hua Wu 0000-0002-9920-3486; Dong-Hui Cao 0000-0002-6795-6395; Zhi-Fang Jia 0000-0001-9838-1726; Ao Shen 0000-0002-3169-0062; Jing Jiang 0000-0001-9714-9255; Xue-Yuan Cao 0000-0002-1982-1672.

Ma YJ

Filipodia

In three simple steps, I eliminated 90 percent of all my blind dates, including one recommended by my old friend Karen. She called one evening to tell me that she had become good friends with a beautiful girl who reminded her of me. She said she knew we would hit it off. Sorry, I said, you re ruled out by rule number two.

Sunrise on the eastern coast is a special event. I stood at Dolphin s Nose, a spur jutting1 out into the Bay of Bengal, to behold2 the breaking of the sun s upper limb over the horizon of the sea. As the eastern sky started unfolding like the crimson3 petals4 of a gigantic flower, I was overcome by a wave of romantic feelings and nostalgia5（，） -- vivid memorie not diminished by the fact that almost ten years had passed.

Ma YJ

1 Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020:GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries.

2021;71:209-249 [PMID:33538338 DOI:10.3322/caac.21660]

2 Wang K, Yuen ST, Xu J, Lee SP, Yan HH, Shi ST, Siu HC, Deng S, Chu KM, Law S, Chan KH, Chan AS, Tsui WY, Ho SL, Chan AK, Man JL, Foglizzo V, Ng MK, Ching YP, Cheng GH, Xie T, Fernandez J, Li VS, Clevers H, Rejto PA, Mao M, Leung SY. Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer.

2014; 46:573-582 [PMID:24816253 DOI:10.1038/ng.2983]

3 Nakajima T, Maekita T, Oda I, Gotoda T, Yamamoto S, Umemura S, Ichinose M, Sugimura T, Ushijima T, Saito D.Higher methylation levels in gastric mucosae significantly correlate with higher risk of gastric cancers.

2006; 15:2317-2321 [PMID:17119066 DOI:10.1158/1055-9965.epi-06-0436]

4 Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M, Arii K, Kaneda A, Tsukamoto T, Tatematsu M,Tamura G, Saito D, Sugimura T, Ichinose M, Ushijima T. High levels of aberrant DNA methylation in Helicobacter pyloriinfected gastric mucosae and its possible association with gastric cancer risk.

2006; 12:989-995 [PMID:16467114 DOI:10.1158/1078-0432.ccr-05-2096]

5 Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma.

1991; 325:1127-1131 [PMID:1891020 DOI:10.1056/nejm199110173251603]

6 Shimazu T, Asada K, Charvat H, Kusano C, Otake Y, Kakugawa Y, Watanabe H, Gotoda T, Ushijima T, Tsugane S.Association of gastric cancer risk factors with DNA methylation levels in gastric mucosa of healthy Japanese:a crosssectional study.

2015; 36:1291-1298 [PMID:26354778 DOI:10.1093/carcin/bgv125]

7 Nakajima T, Enomoto S, Yamashita S, Ando T, Nakanishi Y, Nakazawa K, Oda I, Gotoda T, Ushijima T. Persistence of a component of DNA methylation in gastric mucosae after Helicobacter pylori eradication.

2010; 45:37-44[PMID:19821005 DOI:10.1007/s00535-009-0142-7]

8 Song CX, Yi C, He C. Mapping recently identified nucleotide variants in the genome and transcriptome.

2012; 30:1107-1116 [PMID:23138310 DOI:10.1038/nbt.2398]

9 Mellén M, Ayata P, Dewell S, Kriaucionis S, Heintz N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system.

2012; 151:1417-1430 [PMID:23260135 DOI:10.1016/j.cell.2012.11.022]

10 Thomson JP, Lempiäinen H, Hackett JA, Nestor CE, Müller A, Bolognani F, Oakeley EJ, Schübeler D, Terranova R,Reinhardt D, Moggs JG, Meehan RR. Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome.

2012; 13:R93 [PMID:23034186 DOI:10.1186/gb-2012-13-10-r93]

11 Park JL, Kim HJ, Seo EH, Kwon OH, Lim B, Kim M, Kim SY, Song KS, Kang GH, Choi BY, Kim YS. Decrease of 5hmC in gastric cancers is associated with TET1 silencing due to with DNA methylation and bivalent histone marks at TET1 CpG island 3'-shore.

2015; 6:37647-37662 [PMID:26462176 DOI:10.18632/oncotarget.6069]

12 Yang Q, Wu K, Ji M, Jin W, He N, Shi B, Hou P. Decreased 5-hydroxymethylcytosine (5-hmC) is an independent poor prognostic factor in gastric cancer patients.

2013; 9:1607-1616 [PMID:23980508 DOI:10.1166/jbn.2013.1713]

13 Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, Malfertheiner P, Graham DY, Wong VWS, Wu JCY, Chan FKL, Sung JJY, Kaplan GG, Ng SC. Global Prevalence of Helicobacter pylori Infection:Systematic Review and Meta-Analysis.

2017; 153:420-429 [PMID:28456631 DOI:10.1053/j.gastro.2017.04.022]

14 Alipour M. Molecular Mechanism of Helicobacter pylori-Induced Gastric Cancer.

2021; 52:23-30[PMID:32926335 DOI:10.1007/s12029-020-00518-5]

15 Elzouki AN, Buhjab SI, Alkialani A, Habel S, Sasco AJ. Gastric cancer and Helicobacter pylori infection in the eastern Libya:a descriptive epidemiological study.

2012; 13:85-88 [PMID:22980598 DOI:10.1016/j.ajg.2012.06.002]

16 Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the hallmarks of cancer.

2017; 357 [PMID:28729483 DOI:10.1126/science.aal2380]

17 Do C, Shearer A, Suzuki M, Terry MB, Gelernter J, Greally JM, Tycko B. Genetic-epigenetic interactions in cis:a major focus in the post-GWAS era.

2017; 18:120 [PMID:28629478 DOI:10.1186/s13059-017-1250-y]

18 Liu H, Xu T, Cheng Y, Jin MH, Chang MY, Shu Q, Allen EG, Jin P, Wang X. Altered 5-Hydroxymethylcytosine Landscape in Primary Gastric Adenocarcinoma.

2019; 38:1460-1469 [PMID:31657619 DOI:10.1089/dna.2019.4965]

19 Zhao R, Liu Z, Xu W, Song L, Ren H, Ou Y, Liu Y, Wang S. Helicobacter pylori infection leads to KLF4 inactivation in gastric cancer through a TET1-mediated DNA methylation mechanism.

2020; 9:2551-2563 [PMID:32017451 DOI:10.1002/cam4.2892]

20 Strand SH, Hoyer S, Lynnerup AS, Haldrup C, Storebjerg TM, Borre M, Orntoft TF, Sorensen KD. High levels of 5-hydroxymethylcytosine (5hmC) is an adverse predictor of biochemical recurrence after prostatectomy in ERG-negative prostate cancer.

2015; 7:111 [PMID:26478752 DOI:10.1186/s13148-015-0146-5]

21 Mariani CJ, Vasanthakumar A, Madzo J, Yesilkanal A, Bhagat T, Yu Y, Bhattacharyya S, Wenger RH, Cohn SL, Nanduri J, Verma A, Prabhakar NR, Godley LA. TET1-mediated hydroxymethylation facilitates hypoxic gene induction in neuroblastoma.

2014; 7:1343-1352 [PMID:24835990 DOI:10.1016/j.celrep.2014.04.040]

22 Tsai YP, Chen HF, Chen SY, Cheng WC, Wang HW, Shen ZJ, Song C, Teng SC, He C, Wu KJ. TET1 regulates hypoxiainduced epithelial-mesenchymal transition by acting as a co-activator.

2014; 15:513 [PMID:25517638 DOI:10.1186/s13059-014-0513-0]

23 Valenzuela-Valderrama M, Cerda-Opazo P, Backert S, González MF, Carrasco-Véliz N, Jorquera-Cordero C, Wehinger S, Canales J, Bravo D, Quest AFG. The

Urease Virulence Factor Is Required for the Induction of Hypoxia-Induced Factor-1α in Gastric Cells.

2019; 11 [PMID:31185594 DOI:10.3390/cancers11060799]

24 Abass A, Okano T, Boonyaleka K, Kinoshita-Daitoku R, Yamaoka S, Ashida H, Suzuki T. Effect of low oxygen concentration on activation of inflammation by Helicobacter pylori.

2021; 560:179-185[PMID:34000467 DOI:10.1016/j.bbrc.2021.04.123]