| Objective: Esophageal squamous cell carcinoma(ESCC)is often in the middle and advanced stage at the time of diagnosis,resulting in its high mortality.Therefore,the early prevention of ESCC is important.Unbalanced dietary structure and nutrient deficiency may be the risk factors of ESCC and its precancerous lesions.For example,studies have shown that B vitamins involved in one-carbon metabolism are likely to be associated with the initiation and development of cancers including ESCC,but the conclusions on the association between vitamin B12 and cancer risk have always been inconsistent.The purpose of this study was to carry out epidemiological study in a high risk area of ESCC in Huai’an,Jiangsu Province to investigate the influencing factors of esophageal precancerous lesions(EPLs).Then,the study aimed to figure out the possible role of vitamin B12 and its regulated epigenetic modification in the early stage of ESCC by using dietary samples,serum biomarkers,genetic polymorphism and epigenetics,so as to contribute to the primary prevention of ESCC and find biomarkers for the early prediction and diagnosis of ESCC.Methods:(1)Based on the Early Diagnosis and Early Treatment Project of Esophageal Cancer(EDETPEC)in Huai’an District,Huai’an City,Jiangsu Province from September 2012 to December 2017,high-risk residents aged 35 to 75 were screened by gastroscopy.A total of 11518 subjects were included,including 667 patients with EPLs.The epidemiological survey was conducted using a well-designed questionnaire,whose contents included basic information,eating habits,food intake frequency,history of gastrointestinal diseases,family history of cancer,smoking,drinking,tea drinking,etc.Unconditional univariate Logistic regression was used and the statistically significant variables were further analyzed by the unconditional multivariate Logistic regression model.Odds ratio and 95% confidence interval were obtained after the adjustment for confounding factors.(2)Based on the EDETPEC from January 2014 to December 2017,a case-control study was carried out.Two hundred patients with mild and moderate dysplasia were randomly selected as the EPL cases,and 200 healthy controls were matched by the same region,the same gender and the age difference within 2 years.Three-day duplicate diet samples were collected for following measurement and analysis.The dietary intake of vitamin B12 and cobalt,the serum levels of vitamin B12,folate,homocysteine(Hcy),transcobalamin II(TC II),S-adenosylmethionine(SAM),5-methyltetrahydrofolate(5-MTHF)and Helicobacter pylori Antibody(HP-Ab)in serum were measured.The genotype of TCN2 C776 G was detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)analysis.Wilcoxon signed rank test and conditional Logistic regression model were used to analyze the differences of vitamin B12-related markers between the two groups and the association with EPL risk.Chi-square test was used to analyze the difference of genotype distribution of TCN2 C776 G between the two groups,and the association between TCN2 C776 G polymorphism and the risk of EPL was analyzed by conditional Logistic regression model.(3)Based on the case-control study,4 samples of EPL cases and 4 samples of matched healthy controls were selected.The differentially methylated Cp G positions(DMP)between the two groups were preliminarily screened and analyzed by Illumina Infinium Methylation EPIC Bead Chip,then 100 pairs of samples were selected and the candidate biomarker genes were verified by targeted bisulfite sequencing(TBS).The relationship between methylation level and vitamin B12-related markers was expressed by Pearson or Spearman correlation coefficient.Chi-square test,Fisher exact test,Wilcoxon signed rank test and conditional multivariate Logistic regression model were used to analyze and integrate the results of TBS.Results:(1)Unconditional multivariate Logistic regression model showed that male,increased age,excessive smoking,passive smoking,drinking liquor,overweight or obesity,irregular diet,hot diet,fried food,pickled food,mildew of stored grains,drinking shallow well water or surface water,and history of gastrointestinal diseases such as gastroenteritis,esophagitis and gastric and duodenal ulcers were significantly associated with the increased risk of EPL(P<0.05),while drinking deep well water,consuming vegetables,fruits,nuts,mushrooms,legumes,animal liver and yeast were significantly associated with the reduced risk of EPL(P<0.05).(2)(1)Conditional multivariate Logistic regression model showed that dietary vitamin B12 intake was not significantly associated with the risk of EPL(Ptrend>0.05);The serum levels of vitamin B12,TC II,folate and dietary intake of cobalt were significantly associated with the reduced risk of EPL(Ptrend<0.05);The serum levels of Hcy and 5-MTHF were significantly associated with the increased risk of EPL(Ptrend<0.05).(2)Serum vitamin B12 level was significantly negatively associated with serum Hcy level(Ptrend<0.05),and positively associated with serum folate level(Ptrend<0.05);Compared with the lowest quartile,the highest quartile of serum SAM level was associated with the increase of serum vitamin B12 level(P<0.05).(3)After subgroup analysis of gender,the negative association between serum vitamin B12 level and the risk of EPL can only found in the male group(Ptrend<0.05),while the female group showed a completely different trend,whereas the trend was not statistically significant(Ptrend>0.05).(4)In the male group,compared with genotype TCN2 776 CC,the risk of EPL in genotype CG significantly increased by 191%(P<0.05),and an insignificant increase trend of EPL risk was also found in the genotype GG(P>0.05).(5)Interaction analysis showed that compared with the CC genotype with highest quartile of serum vitamin B12 level,the EPL risk of CG genotype with the second highest quartile and the lowest quartile of serum vitamin B12 level increased by 19 times and 50 times,respectively(P<0.05),the EPL risk of CG genotype with the third highest quartile and the second quartile of serum vitamin B12 level increased by 16 and 18 times,respectively(P<0.05).(3)(1)Illumina Infinium Methylation EPIC Bead Chip showed that the methylation level of EPL samples were more likely to be within the range of 0.2~0.8,while the sites of healthy controls tended to be hypermethylated or hypomethylated.There were dramatic differences in methylation between the two groups.The global DNA methylation level in healthy controls was significantly higher than that in EPL cases(P<0.05).(2)The serum level of vitamin B12 was significantly negatively correlated with the lower quartile of methylation level,and the serum level of Hcy was significantly negatively correlated with the median of methylation level(P<0.05).(3)Compared with the healthy controls,69.0% of the DMP were down-regulated and 31.0% of the DMP were up-regulated in the EPL group.(4)By TBS and conditional multivariate Logistic regression analysis,the methylation levels of UGT2B15 and FGFR2 gene promoters were significantly associated with the increased risk of EPL(P<0.05).Conclusion:(1)In the high risk area in Huai’an,EPL shows a distinct epidemiological pattern.Smoking and drinking may play a minor role,while environmental exposure,unhealthy eating habits and imbalanced diet may play the dominant role in the initiation of ESCC.(2)Although there was no significant difference in dietary intake of vitamin B12 between EPL cases and healthy controls,the serum level and transport and utilization efficiency of vitamin B12 in healthy controls were significantly higher than those in EPL cases.Vitamin B12 may play a protective role in the early stage of initiation of ESCC through one-carbon metabolism pathway.The TCN2 C776 G polymorphism was also related to the susceptibility to EPL,and its polymorphism may interact with the nutritional status of vitamin B12 to influence the risk of EPL.(3)In EPL cases,there may be aberrant DNA methylation,in which the global DNA methylation level decreases and the methylation level of specific Cp G island aberrantly increases.For example,the gene promoter regions of UGT2B15 and FGFR2 were abnormally hypermethylated,and the mechanism may be related to the methionine cycling involved by vitamin B12.In addition,the methylation in the promoter regions of the above two genes may be promising molecular markers in the early prediction and diagnosis of ESCC. |
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