Application of choline to prevention of digestive system tumors

Abstract

The invention relates to application of a product containing choline products to prevention of digestive system tumors of PEMT gene polymorphic site rs7946CC type carriers. According to the invention, when a detected object is a PEMT rs7946CC carrier, it is predicted that the digestive system tumor disease risk of the detected object is high; a PEMT rs7946CC type carrier uses choline products, so that the reduction of the disease risk of digestive system tumors of the PEMT rs7946CC type carrier is facilitated.

Description

Technical Field

[0001] The present invention relates to the use of choline-containing products for preventing digestive system tumors in carriers of the rs7946CC genotype of the PEMT gene polymorphism locus. Background Art

[0002] Digestive system tumors are the most aggressive tumor subtypes, with the highest mortality rate, posing a huge threat to the lifespan of the population and becoming a huge burden on the medical system and social economy. The main reason for the poor prognosis of digestive system tumors is the lack of timely and accurate diagnosis. Therefore, exploring new biomarkers from multiple dimensions may contribute to its early diagnosis and prevention. The etiology of cancer is complex, and its pathogenesis has not been fully elucidated yet. However, the generally recognized view is that tumors may be caused by the combined action of genetic and environmental susceptibility factors. The importance of one-carbon metabolism (OCM) in tumors has been recognized more than 70 years ago. The disorder of OCM may potentially change the body's susceptibility to tumors by causing abnormal DNA synthesis and DNA methylation. Therefore, in recent decades, the role of various elements involved in the OCM pathway in carcinogenesis has been strongly concerned by researchers.

[0003] Choline is another source of methyl groups in cellular methylation reactions because choline can participate in OCM through its only oxidation product, betaine. The abnormal state of choline metabolism is related to malignant transformation. Phosphatidylethanolamine N-methyltransferase (PEMT, EC 2.1.1.17) is a gene related to choline metabolism. The enzyme encoded by this gene catalyzes the only de novo choline synthesis reaction in the body. Specifically, under the catalysis of PEMT, with SAM as the methyl donor, phosphatidylethanolamine is methylated to generate phosphatidylcholine (PC). Under normal conditions, the PEMT pathway accounts for 20-30% of the total phosphatidylcholine synthesis, and phosphatidylcholine can further regenerate a considerable amount of choline. The PEMT enzyme catalyzes the de novo choline synthesis reaction, and choline can be further oxidized to generate betaine. Betaine serves as an alternative methyl donor in the one-carbon metabolism pathway, which can remethylate homocysteine to methionine and ultimately generate S-adenosylmethionine, which is the general methyl donor for most methylation reactions, including DNA methylation. Choline and betaine are important factors in the one-carbon metabolism pathway. They are closely related to the folate cycle and interact complexly. Therefore, they may participate in DNA synthesis and repair, thereby affecting the occurrence and formation of tumors.

[0004] The PEMT gene has a high degree of polymorphism. One of its functional polymorphic sites, rs7946, has been reported to be associated with breast cancer risk and has an interaction with choline intake level and betaine intake level. However, the biological mechanism involved lacks relevant research and reports and thus has not been well understood. In addition, no research data elaborating on the potential association between choline and digestive system tumors have been found. Summary of the Invention

[0005] To overcome the above-mentioned defects in the prior art, one object of the present invention is to provide a use of a choline-containing product for preventing the occurrence of digestive system tumors based on the genotype of the PEMT rs7946 polymorphic site.

[0006] Use of a choline-containing product for preventing digestive system tumors in carriers of the CC genotype at the PEMT gene polymorphic site rs7946. In the present invention, when the test subject is a carrier of the PEMT rs7946 CC genotype, the risk of suffering from digestive system tumors in the test subject is predicted to be high. The use of a choline-containing product by carriers of the PEMT rs7946 CC genotype helps reduce the risk of suffering from digestive system tumors in such carriers.

[0007] In the use provided by the present invention, when the serum choline / betaine ratio in carriers of the PEMT rs7946 CC genotype is low, the risk of carriers of the CC genotype suffering from digestive system tumors increases. As a preference, when the choline / betaine ratio < 0.412, the risk of carriers of the CC genotype suffering from digestive system tumors increases significantly.

[0008] In the use provided by the present invention, the amount of choline chloride in the choline-containing product is 100 mg - 3 g.

[0009] In the use provided by the present invention, the choline-containing product includes choline chloride, lecithin, phosphatidylcholine, etc.

[0010] The use of a choline-containing product provided by the present invention for preventing digestive system tumors in carriers of the PEMT gene polymorphic site rs7946 CC genotype can effectively prevent the occurrence and progression of digestive tract tumors in carriers of the PEMT gene polymorphic site rs7946 CC genotype, and has unexpected effects.

[0011] The following further describes the embodiments of the present invention in conjunction with examples. Detailed Embodiments

[0012] Example 1: Analysis of the correlation between PEMT rs7946 and digestive tract tumors

[0013] Screening and pairing of the study population and cases-controls

[0014] This study was conducted in the cohort of the China H-type Hypertension Registry Study (CHHRS, URL: http: / / www.chictr.org.cn / showprojen.aspx?proj=28262; Clinical Registration Number: ChiCTR1800017274; Registration Date: July 20, 2018). The enrollment of subjects and the collection of baseline information for this study were completed in 2016, with a total of 234,714 participants from Lianyungang and Rongcheng. Subsequently, according to the study protocol, follow-up was conducted every 3 months until 2019. To maximize the benefit / cost, this study adopted a nested case-control study design. During the follow-up period, a total of 2,021 newly diagnosed cancer patients were collected. The control group was selected from the 232,355 participants who were still alive at the end of the study, and these participants had no occurrence of any tumors before participating in the study and throughout the follow-up period. Matching was performed at a case-control ratio of 1:1, and the matching factors included age (±1 year), gender, center, and place of residence at the time of enrollment. After excluding participants with missing genotype data or unmatched cases or controls (n = 148), 1,947 pairs of digestive system tumor cases-controls were obtained. After excluding 1,196 pairs of non-digestive system tumor patients, finally, 751 pairs of digestive system tumor cases-controls with complete PEMT rs7946 genotype data were selected for the final data analysis.

[0015] Genotyping of PEMT rs7946

[0016] In this study, the DNA used for genotyping was from venous blood leukocytes.

[0017] Quantitative determination of betaine and choline contents in serum / plasma using LC-MS / MS

[0018] After the samples were pretreated such as protein removal, chromatographic separation of the sample analytes was achieved using a liquid chromatography column, and they reached the mass spectrometry detector at different retention times to obtain the corresponding mass spectrometry diagrams. A standard curve was made by the ratio of the peak area to the peak area of the internal standard corresponding to the solution concentration, so as to calculate the concentration contents of betaine and choline in the samples to be measured.

[0019] Statistical analysis

[0020] Normally distributed variables were expressed as mean ± standard deviation (SD), and paired t-tests were used for comparison. Non-normally distributed variables were expressed as median (interquartile range, IQR), and rank sum tests were used for comparison. Categorical variables were expressed as n (%), and chi-square tests were used for comparison.

[0021] The relationship between the polymorphic locus PEMT rs7946 and the risk of digestive system tumors was evaluated using the odds ratio and 95% confidence interval of the conditional logistic regression model. All regression analysis models were adjusted for possible confounding variables of the relationship between polymorphism and tumors, as well as variables clearly identified as tumor risk factors (including smoking and drinking status). When testing the potential interaction between PEMT rs7946 and major relevant covariates through stratified analysis, unconditional logistic regression was used, and the adjusted variables in the adjusted model also included matching variables.

[0022] In all analyses, a two-tailed test was considered statistically significant. All statistical analyses were performed using R software version 4.0.5 (http: / / www.R-project.org / ).

[0023] Results

[0024] Relationship between PEMT rs7946 and the risk of digestive tract tumors

[0025] As shown in Table 1-1, in the conditional logistic regression model, the risk of digestive system tumors in patients with the CC genotype increased by 31% (adjusted OR (odds ratio): 1.31; 95% CI (confidence interval): 1.04, 1.66, P = 0.023), and this difference was statistically significant.

[0026] Table 1-1. Correlation between PEMT rs7946 genotype and the risk of digestive system tumors (a total of 751 pairs)

[0027]

[0028] Using conditional logistic regression, the adjusted model adjusted for educational level, smoking status, drinking status, mean systolic blood pressure, BMI, total cholesterol, triglyceride, fasting blood glucose, Hcy, vitamin B12, folic acid, choline, betaine, and choline / betaine ratio.

[0029] Mediation effect analysis

[0030] In this study population, when comparing the choline and betaine levels between the CC genotype and the TT / CT genotype at the PEMT rs7946 polymorphism locus, it was found that the choline level of CC genotype carriers was lower than that of TT / CT genotype carriers, but the difference did not reach statistical significance (p = 0.080); however, the betaine level of CC genotype carriers was significantly higher than that of TT / CT genotype carriers (p = 0.040); correspondingly, compared with the TT / CT genotype, the choline / betaine ratio in the CC genotype population was lower, and this difference was statistically significant (p = 0.001). Subsequently, in order to test whether choline, betaine levels, and the choline / betaine ratio might be mediating factors in the association between the PEMT rs7946 polymorphism locus and the risk of digestive system tumors, this section used the bootstrap test to test for potential mediating effects. The results showed that although neither choline nor betaine alone showed a mediating effect on the above-mentioned association, the choline / betaine ratio showed a significant mediating effect (P = 0.018, mediating effect proportion 13.55%. Tables 1-2, Table 1-3).

[0031] Table 1-2. Effects of different PEMT rs7946 genotypes (TT and CT genotypes combined) on serum choline, betaine concentrations, and choline / betaine ratio

[0032]

[0033] For independent samples, the Wilcoxon rank-sum test was used

[0034] Table 1-3. Mediation analysis of serum choline, betaine, and choline / betaine ratio

[0035]

[0036] Using the bootstrap test, the model adjusted for smoking status, drinking status, homocysteine, vitamin B12, and total folic acid. The results of the stratified analysis suggested a significant gene-environment interaction between PEMT rs7946 and the choline / betaine ratio: among participants with a lower serum choline / betaine ratio (<0.412), the risk of digestive system tumors in CC genotype carriers increased by 64% (P 交互 = 0.020).

[0037] Example 2: Preventive effect of choline on N-methyl-N'-nitro-N-nitrosoguanidine-induced digestive tract tumors in rats

[0038] Animals: 6-week-old male Wistar rats with an average body weight of 143.3 ± 12.6 g were housed at a room temperature of 22°C and a relative humidity of 55% ± 5%. A light / dark cycle was maintained every 12 h. The rats were fed a mixed diet and had free access to food and water. They were allowed to acclimatize to the new environment for one week.

[0039] Model establishment: Ten rats were randomly selected as the blank control group. The remaining rats were randomly divided into a model control group, a choline chloride group 1, and a choline chloride group 2. Blank control group: fed with a mixed diet and pure water; Model control group: fed with a mixed diet and given 100 mg / L N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water. The average consumption of MNNG was 20 ml per rat per day. After continuous feeding for 30 weeks, the drinking water was changed to pure water; Choline 1 group: fed with a diet containing choline chloride and given 100 mg / L MNNG in drinking water. The average intake of choline chloride was 10 mg / kg body weight. After continuous feeding for 34 weeks, the average consumption of MNNG was 20 ml per rat per day. After continuous feeding for 30 weeks, the drinking water was changed to pure water; Choline 2 group: fed with a diet containing choline chloride and given 100 mg / L MNNG in drinking water. The average intake of choline chloride was 300 mg / kg body weight. After continuous feeding for 34 weeks, the average consumption of MNNG was 20 ml per rat per day. After continuous feeding for 30 weeks, the drinking water was changed to pure water. At the end of the 34-week experiment, all rats were anesthetized with chloral hydrate, dissected to remove the stomach, and sacrificed. The stomach was opened along the greater curvature, and gross specimens were observed. After grouping and photographing, the specimens were fixed with formaldehyde. The fixed specimens were cut into 3 - 4 pieces along the longitudinal axis, embedded in paraffin, and stained with HE for histological diagnosis. The diagnosis of gastritis was referred to the New Sydney System criteria.

[0040] Calculation of tumor volume:

[0041] For gross specimens, the length and width of the tumor tissue were measured respectively, and the approximate volume of the tumor was calculated according to the formula (length * width 2 ) / 2.

[0042] Statistical analysis: The SAS software was used. The chi-square test was applied to analyze the tumor incidence. The comparison of tumor volumes was analyzed by analysis of variance. 2

[0043] Table 2-1 Effects of choline on the incidence of gastric mucosal lesions (%) and tumor volume

[0044]

[0045] Compared with the blank control group, # P < 0.05, ## P < 0.01; compared with the model group, * P < 0.05, ** P < 0.01.

[0046] Results:

[0047] In addition to carcinogenesis, mucosal inflammation, ulcers, and dysplasia were also observed in the rats of the model group, showing a very significant difference compared with the blank control group. Inflammation and dysplasia were also present in the rats of choline group 1 and choline group 2, showing no significant difference compared with the model group; however, the number of rats with ulcers was significantly less than that in the model group; the number of rats with tumors was also significantly less than that in the model group; the average tumor volume was significantly lower than that in the model group. This indicates that choline within the dose range of 10 mg / kg - 300 mg / kg is helpful in reducing the tumor incidence rate in rats.

Claims

1. The use of products containing choline products for preventing digestive system tumors in carriers of the PEMT gene polymorphism site rs7946 CC type.

2. The use according to claim 1, characterized in that The PEMT rs7946 had a lower serum choline / betaine ratio in vivo.

3. The use according to claim 2, characterized in that: The choline / betaine ratio is <0.

412.

4. The use according to claim 1, characterized in that The choline-containing product contains 100 mg to 3 g of choline chloride.

5. The use according to claim 1, characterized in that: The choline products include choline chloride, lecithin and phosphatidylcholine and the like.

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