The Interaction Among MTHFR Rs1801133 Polymorphism,NAFLD And H.pylori Infection In The Levels Of CEA,CA19-9,CA72-4 And Modeling Gastrointestinal Tumorigenesis Using The Organoids Derived From CiMICs

The global incidence of gastrointestinal malignancies accounts for more than 50%of adult malignancies.Due to the lack of specific clinical manifestations in the early stage of gastrointestinal malignancies,patients are mostly in the advanced or late stage when they see a doctor due to gastrointestinal symptoms,which not only affects the survival rate of patients after treatment,but also has a great impact on the quality of life of patients.Therefore,it is necessary to carry out early screening of gastrointestinal malignant tumors in order to achieve the purpose of early diagnosis and early treatment.As a lifestyle disease regulated by multiple factors and interacted by genetic and environmental factors,gastrointestinal malignancies are closely related to Helicobacter pylori(H.pylori)infection.Non-alcoholic fatty liver disease(NAFLD)as the liver manifestation of metabolic syndrome,can increase the risk of gastrointestinal tumors by affecting intestinal flora,aggravating insulin resistance and lipid metabolism disorder.As a key enzyme in folate metabolism,MTHFR plays an important role in maintaining the structure and function of DNA.When the cytosine(C)at position rs1801133 of MTHFR is replaced by Thymine(T),which will affect the stability of single strand and double strand DNA and DNA methylation,and induce the occurrence of gastrointestinal malignant tumors.At present,the research results on the susceptibility and correlation between MTHFR rs1801133 polymorphism and NAFLD and H.pylori infection are inconsistent.Meanwhile,there are few studies on the relationship between H.pylori infection complicated with NAFLD and MTHFR rs1801133 gene polymorphism and gastrointestinal tumor markers(GTM).In order to further explore the relationship between MTHFR and gastrointestinal tumorigenesis,the selection of cell lines that meet the experimental conditions is the basis of the experimental work.It is very important to select cell lines that can modeling the characteristics of disease in vitro and vivo.Intestinal cancers are developed from intestinal epithelial stem cells(ISCs)in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes.ISCs play a key role in maintaining the homeostasis of gut epithelium.In 2009,Sato et al established a three-dimensional(3D)culture system,which mimicked the niche microenvironment by employing the niche factors,and successfully grew crypt ISCs into organoids or Mini-guts in vitro.Since then,the intestinal organoid technology has been used to delineate cellular signaling in ISC biology.However,the cultured organoids consist of heterogeneous cell populations,and it was technically challenging to introduce genomic changes into 3D organoids.Thus,there was a technical necessity to develop a two-dimensional(2D)ISC culture system for effective genomic manipulations.PART1 THE RELATIONSHIP AND INTERACTION AMONG MTHFR RS1801133 POLYMORPHISM,NON-ALCOHOLIC FATTY LIVER DISEASE AND GASTROINTESTINAL TUMOUR MARKERSObjective:(1)To analyze the clinical features of NAFLD;(2)To investigate the relationship between NAFLD and MTHFR rs1801133 gene polymorphism;(3)To statistically analyze the relationship and interaction among MTHFR rs1801133 polymorphism,NAFLD and GTM:Carcinoma embryonic antigen(CEA)?Carbohydrate antigen 19-9(CA19-9)?Carbohydrate antigen 72-4(CA72-4).Methods:1023 subjects were health checked-up in the urban of Chongqing from January 2016 to October 2020,including 425 cases in NAFLD group and598 Cases in control group.Sanger sequencing method was used to screen MTHFR rs1801133 gene polymorphism,and electrochemiluminescence method was used to examine the GTM of subjects:CEA,CA19-9,CA72-4.The risk factors of NAFLD were analyzed by logistic regression;Hardy-Weinberg equilibrium of MTHFR rs1801133 polymorphism was calculated by chi-square goodness of fit test.Forward logistic regression was used to calculate odds ratio(OR)and 95%confidence interval(CI)to evaluate the relationship among NAFLD and risk factors;Multifactor dimensionality reduction(MDR)was used to analyze gene-environment interactions;General linear model was used to test the interaction among MTHFR rs1801133 polymorphism,NAFLD and GTM(CEA,CA19-9 and CA72-4).P<0.05 was considered statistically significant.Result:(1)The prevalence of NAFLD in male(50.7%)was significantly higher than that in female(23.5%;P<0.001).The body mass index(BMI)of NAFLD group was 26.42�2.88,which was significantly higher than that of control group 23.05�2.730(P<0.001).With the increase of BMI,the prevalence of NAFLD increased(BMI<18.5,underweight(UW):5%;BMI18.5?23.9,normal weight(NW):17.4%;BMI 24.0?27.9,overweight(OW):54.8%;BMI>28.0,obesity(OB):82.4%,P<0.001).In NAFLD group,hypertension:diastolic blood pressure(DBP)and systolic blood pressure(SBP)increased;Liver function damage:gamma glutamyltransferase,?-GT),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)increased;Dyslipidemia:serum total cholesterol(s TC),serum triglyceride(s TG),low density lipoprotein cholesterol(LDL-C)increased and high density lipoprotein cholesterol(HDL-C)decreased.The prevalence of eosinophils(EO)was higher than that in the control group(P<0.05).The levels of white blood cell(WBC),red blood cell(RBC),hemoglobin(HB)and hematocrit value(HCT)in NAFLD group were higher than those in control group(P<0.05).The median CEA level(P25,P75)of NAFLD group was 1.90(1.20,2.90)ng/ml,which was higher than that of control group(1.20,2.50)ng/ml(P=0.005);However,there was no significant difference in the levels of CA19-9 and CA72-4 between the two groups(P>0.05).(2)The frequency of T allele(47.9%)was higher than that of C allele(37.5%)in NAFLD group.C allele frequency(62.5%)was higher than T allele frequency(52.1%,P<0.001)in the control group.In NAFLD group,the OR value and 95%CI of MTHFR rs1801133 polymorphism decreased in the order of TT genotype>CT genotype>CC genotype;the OR value and95%CI of MTHFR rs1801133 recessive model(TT vs.CC+CT genotype)were higher than those of the dominant model(CC vs.CT+TT genotype),which indicated that different genotypes had different susceptibility to NAFLD(P<0.05).(3)Logistic multivariate regression analysis showed that BMI,high SBP,high?-GT,high ALT,high s TG,low HDL-C,high LDL-C,WBC level,high EO and MTHFR rs1801133 polymorphisms were risk factors for NAFLD(P<0.05).(4)MDR modeling analysis showed that there may be environment-environment interaction between BMI and HDL-C and NAFLD.There may be gene-environment interaction between MTHFR RS1801133 polymorphism,BMI and HDL-C and NAFLD.The dendrogram suggested that BMI and?-GT,MTHFR rs1801133polymorphism and HDL-C all had synergistic effects on the incidence of NAFLD,and BMI and?-GT had stronger synergistic effects.(5)The CEA level of MTHFR rs1801133 TT genotype was higher than that of MTHFR rs1801133 CC and CT genotype,and the CEA level of TT genotype,CT genotype and CC genotype showed a decreasing trend;The lines between the CEA mean levels of the three genotypes of NAFLD and control group were not parallel,which suggesting that NAFLD and Mt HFR rs1801133 polymorphism had an interaction effect on CEA levels.Conclusion:(1)Obesity,hypertension,hyperlipidemia and abnormal liver function were common risk factors of NAFLD in the clinical feature which suggested that chronic metabolic disease was a risk factor for NAFLD;(2)After adjusting for related factors,MTHFR rs1801133polymorphism was a risk factor for NAFLD.T allele mutation increases the risk of NAFLD.Overweight(OW)/Obesity(OB),high?-GT,MTHFR rs1801133 polymorphism and low HDL-C all had synergistic effects on NAFLD;(3)MTHFR rs1801133 polymorphism and NAFLD may had an interactive effect on the increase of CEA level.The mutation of MTHFR RS1801133 T allele and NAFLD may lead to the elevation of CEA level.PART2 THE RELATIONSHIP AND INTERACTION AMONG MTHFR RS1801133 POLYMORPHISM,HELICOBACTER PYLORI INFECTION AND GASTROINTESTINAL TUMOR MARKERSObjective:(1)To investigate the clinical characteristics of H.pylori infection in Han population in Chongqing;(2)To analyze the relationship between H.pylori infection and MTHFR rs1801133 polymorphism in Han population in Chongqing;(3)To statistically analyze the relationship and interaction among H.pylori infection,MTHFR rs1801133 gene polymorphism and gastrointestinal tumour markers(CEA,CA19-9 and CA72-4).Methods:A total of 152 subjects,including 58 cases of ¹³C-UBT positive group and 94 cases of ¹³C-UBT negative group,were all Han people who completed health check-up in urban of Chongqing from January 2016 to October 2020.All subjects completed the Carbon 13 Urea Breath Test(¹³C-UBT),MTHFR rs1801133 polymorphism screening and GTM(CEA,CA19-9 and CA72-4)tests.Subjects were grouped according to ¹³C-UBT results.DOB value?4 was defined as ¹³C-UBT positive,which was H.pylori infection.DOB value<4 was defined as ¹³C-UBT negative and neither H.pylori infection.Chi-square test was used to analyze the difference of count data,c²test or Fisher exact test was used to analyze the difference of measurement data,andc²test was used to compare the classification variables between groups.The genotype frequencies of MTHFR rs1801133 polymorphism were analyzed by logistic regression.LR logistic regression was used to calculate OR and 95%CI to evaluate the relationship between ¹³C-UBT positive and risk factors.Multivariate analysis of variance was used to analyze the interaction among MTHFR rs1801133 polymorphism and GTM,¹³C-UBT results.Bootstrap method was used to calculate 95%CI,and logistic regression was used to calculate the statistical interaction between MTHFR rs1801133 gene polymorphism and classified variables and continuous variables in ¹³C-UBT positive risk factors.Result:(1)Among the Han people in Chongqing,the positive rate of ¹³C-UBT in male(43.4%)was higher than that in female(26.1%,P=0.044);the positive rate of ¹³C-UBT in age>60 group(71.4%)was significantly higher than that in other age group(age<40:36.4%;age 40?60:34.5%,P=0.027);the levels of CEA 2.08�1.04ng/ml,CA72-4 3.70(1.60,5.93)U/ml and WBC6.00�1.34 10^{^9}/L in ¹³C-UBT positive group were higher than those in¹³C-UBT negative group(CEA:1.69�0.74ng/ml,P=0.008;CA72-4:2.10(1.08,4.23)U/ml,P=0.004;WBC:5.54�1.42 10^{^9}/L,P=0.045);in MTHFR rs1801133 recessive model(TT vs.CC+CT),the positive rate of ¹³C-UBT in TT genotype(57.7%)was higher than that in CC+CT genotype(34.1%,P=0.028).(2)Logistic regression analysis showed that DBP,CEA,CA72-4 and MTHFR rs1801133 recessive model(TT vs.CC+CT genotype)were risk factors for ¹³C-UBT positive(P<0.05).(3)By analyzing the additive interactions effect between MTHFR rs1801133 polymorphism and CEA on ¹³C-UBT results,MTHFR rs1801133 polymorphism had negative additive interaction with CEA levels(P=0.026).The Relative Excess Risk of Interaction(RERI)was-1.407(95%CI=-3.737?-0.277),Synergy Index(S)was 0.468(95%CI=0.072?0.910),Attributable Proportion(AP)was 63.1%(95%CI=-1.941?-0.079).(4)After adjusting for other factors,the level of CEA in male(2.13�0.11 ng/m L)was significantly higher than that in female(1.66�0.14ng/ml,P=0.002).CEA level in age>60 years group(2.41�0.22 ng/m L)was significantly higher than that in other groups(age<40:1.51�0.18 ng/ml;age 40?60:1.78�0.09,P=0.009).When ¹³C-UBT was positive,T allele decreased CEA level;when ¹³C-UBT was negative,T allele increased CEA level(P=0.004).The level of CA19-9 in female(15.63�2.30 U/m L)was significantly higher than that in male(9.25�1.84U/m L,P=0.010).The level of CA72-4 in ¹³C-UBT positive group(5.85�0.93U/m L)was significantly higher than that in negative group(3.18�0.92 U/m L,P=0.020).Conclusion:(1)The infection of H.pylori in Han population in Chongqing was related to gender and age,male has more chances to infect H.pylori than female;The infection rate of H.pylori increased with the increase of age.Meanwhile,H.pylori infection resulted in increased levels of CEA,CA72-4and WBC.(2)MTHFR rs1801133 polymorphism might be a susceptibility factor for H.pylori infection in Han population in Chongqing.And the MTHFR rs1801133 TT genotype might increase the risk of H.pylori infection in Han population in Chongqing area.(3)MTHFR rs1801133 polymorphism had a negative additive interaction with CEA level in H.pylori infection,suggesting that H.pylori infection with different genotypes would lead to different degrees of CEA level.There was a statistical interaction between MTHFR rs1801133polymorphism and ¹³C-UBT results at CEA level.PART3 THE RELATIONSHIP AND INTERACTION AMONG MTHFR RS1801133 POLYMORPHISM,H.PYLORI INFECTION COMPLICATED WITH NON-ALCOHOLIC FATTY LIVER DISEASE AND GASTROINTESTINAL TUMOUR MARKERSObjective:(1)To investigate the clinical characteristics of H.pylori infection and H.pylori infection complicated with NAFLD;(2)To investigate the relationship between H.pylori infection,H.pylori infection complicated with NAFLD and MTHFR rs1801133 gene polymorphism;(3)To statistically analyze the relationship and interaction among H.pylori infection,H.pylori infection complicated with NAFLD,MTHFR rs1801133 gene polymorphism and GTM(CEA,CA19-9 and CA72-4).MDR modeling was used to analyze the interaction between MTHFR RS1801133 recessive model(TT vs.CC+CT genotype)and NAFLD on abnormal GTM levels in h.pylori infected population.Methods:A total of 130 patients were included in the study,including 72 in the control group(without H.pylori infection and NAFLD),30 in the disease group 1(H.pylori infection group)and 28 in the disease group 2(H.pylori infection+NAFLD group).All subjects underwent ¹³C-UBT,abdominal color doppler ultrasound,CEA,CA19-9 and CA72-4 examinations.The measurement data were analyzed by t-test or rank sum test;The counting data were analyzed byc²test or Fisher exact test;The trend of measurement and counting data was analyzed by analysis of variance.The distribution of MTHFR rs1801133 polymorphism among the three groups was analyzed by ordered loistic regression.Univariate and multivariate logistic regression were used to analyze the correlation among human body indexes,blood biochemical examination and MTHFR rs1801133polymorphism with disease.Multivariate logistic regression was used to screen the variables.The differences and interactions among the three groups were analyzed by analysis of variance.MDR modeling was used to analyze gene-environment interaction.Result:(1)Compared with the control group,BMI,blood pressure(DBP and SBP),CEA,CA72-4,?-GT,ALT,s TG and WBC levels were increased in disease group 1 and disease group 2,while HDL-C levels were decreased(P<0.05).The increase levels of BMI,blood pressure(DBP and SBP),CA72-4,?-GT,ALT,s TG and WBC and the decrease level of HDL-C in disease group 2 were significantly higher than those in disease group 1(P<0.05).Compared with the control group,BMI,blood pressure(DBP and SBP),CEA,CA72-4,?-GT,ALT,s TG and WBC levels were increased and HDL-C levels were decreased in disease group 1 and disease group 2(P<0.05).Among them,blood pressure(DBP and SBP),CA72-4,?-GT,ALT,s TG and WBC levels increased(P<0.05)and while HDL-C level decreased(P<0.05)with H.pylori infection and combination of NAFLD.(2)Different MTHFR rs1801133 genotypes had different susceptibility to H.pylori infection and NAFLD,showing a trend of TT genotype>CT genotype>CC genotype.(3)Through logistic univariate and multivariate regression analysis,it was found that SBP,CEA,CA72-4,WBC level and MTHFR rs1801133recessive model(TT vs.CC+CT genotype)were the risk factors of H.pylori infection and H.pylori infection complicated with NAFLD(P<0.05).(4)After adjusting for other factors,the levels of CEA and CA72-4among the three groups were statistically significant(P<0.05);The level of CA72-4 in different genotypes was also statistically significant(P<0.05).With the mutation of MTHFR rs1801133 T allele,the level of CA72-4increased accordingly(P=0.003).There was interaction between control group,disease group 1 and disease group 2 with MTHFR RS1801133polymorphism at CEA(P=0.028)and CA72-4(P<0.001)levels.CEA and CA72-4 levels of MTHFR RS1801133 TT genotype in disease group 2were significantly higher than those in control group and other genotypes in disease group 1.WBC level was increased with the increase of CA19-9 and CA72-4 levels(P<0.05).5)MDR analysis showed that three-dimensional model(MTHFR rs1801133 recessive model,NAFLD,WBC)was the best model.MTHFR rs1801133 recessive model(TT vs.CC+CT genotype)and two-dimensional model(MTHFR rs1801133 recessive model,NAFLD)were efficient models.It is suggested that MTHFR rs1801133 TT genotype and MTHFR rs1801133 CC+CT genotype have gene interaction on the increase of CA72-4 level;in the two-dimensional and three-dimensional models,MTHFR rs1801133 recessive model has gene-environment interaction with NAFLD and WBC.The dendrogram suggests that MTHFR rs1801133 recessive model and NAFLD have a stronger synergistic effect on the increase of CA72-4 level.Conclusion:(1)H.pylori infection might affect the ability of liver lipid metabolism;Inflammation was closely related to H.pylori infection and might affect the occurrence and development of NAFLD.(2)MTHFR rs1801133 T allele mutation was a susceptibility factor for H.pylori infection and H.pylori infection complicated with NAFLD.And MTHFR rs1801133 TT genotype has a higher risk of two diseases.(3)There was interaction between different disease groups and MTHFR rs1801133 polymorphism at CEA and CA72-4 levels.Mutation of MTHFR rs1801133 T allele not only increases the risk of H.pylori infection and NAFLD,but also may affect the elevation of CEA and CA72-4 after disease.(4)Different MTHFR rs1801133 genotypes,NAFLD and inflammatory reaction will have different effects on the increase of CA72-4level in H.pylori infected population.Among them,the synergistic effect of MTHFR rs1801133 TT genotype and NAFLD on the increase of CA72-4 level in H.pylori infected population was the most obvious.PART 4 MODELING GASTROINTESTINAL TUMORIGENESIS USING THE ORGANOIDS DERIVED FROM CONDITIONALLY IMMORTALIZED MOUSE INTESTINAL CRYPT CELLS(CIMICS)Objective:(1)Established a conditionally immortalized mouse intestinal crypt(ci MIC)cell line;(2)To verify the biological characteristics of ci MICs.To verify whether ci MICs can maintain the ability to form intestinal organoids in 3D culture conditions;(3)The activated oncogenes were introduced into ci MICs to model gastrointestinal tumors and verify their tumorigenic,pathological and histological characteristics in vivo.Methods:we established a conditionally immortalized mouse intestinal crypt(ci MIC)cell line by using a piggy Bac transposon-based SV40 T antigen expression system.The biological characteristics and proliferative activity of ci MICs were verified.ci MICs cultured in 2D conditions were further cultured in 3D conditions to verify whether Mini-guts or crypt organoids of intestinal epithelial structure could be formed.By constructing a common mutation of constitutive activation mutation?-catenin and KRASG12D in piggy Bac vector with deletion of n-terminal destruction domain.Using these vectors,we generated 5 stable ci MICs lines.Result:(1)To immortalize primary mouse intestinal crypts(MICs),we transfected the cells with p MPH86 vector and infected with Ad PBase with high efficiency.Stably transposed and immortalized cells were obtained upon hygromycin(Hyg)selection and could be passaged for generations.(2)ci MICs lacked mesenchymal origin cells and could maintain long-term proliferative activity under 2D culture conditions,while retaining the biological characteristics of intestinal epithelial stem cells.(3)ci MICs could form intestinal organoids in 3D culture conditions.And it has many similarities with APC^min+/-mouse derived organoids.(4)Using these vectors,we generated stable ci MICs lines,including ci MIC-FGL(expressing firefly luciferase,GFP and Lac Z),ci MIC-RFP(expressing RFP),ci MIC-mut BC(expressing active mutant?-catenin),ci MIC-mutkras(expressing mutant KRAS),and ci MIC-mut BC/KRAS(expressing both mutant b-catenin and mutant KRAS).Under 3D culture conditions,ci MICs,ci MIC-mut BC,ci MIC-mut KRASand ci MIC�mut BC/KRAS all formed intestinal organoid structures with similar efficiency.(5)In vivo,no mass block was formed at the injection site of ci MIC-FGL and ci MIC-mut BC mixed with PBS;The bioluminescence signal at the injection site of ci MIC-FGL/PBS lasted for 2 weeks,and the bioluminescence signal could not be detected after 7 weeks.ci MIC-mut BC and ci MIC-mut KRAS mixed with ENR medium or Matrigel can form tumors subcutaneously in nude mice.The tumor was easily obtained from the ci MIC-mut BC/ENR injection site,while only small mass was obtained from the ci MIC-FGL/Matrigel injection site mixed with Matrigel.No tumor was detected at the injection site of ci MIC-FGL/ENR medium.(6)In the simulated intestinal tumorigenesis model experiment,the tumor bodies of ci MIC-mut BC and ci MIC-mut KRAS injection groups showed high proliferation and intestinal adenoma like pathological and histological characteristics.Conclusion:(1)Using a piggy Bac transposon-based SV40 T antigen expression system,could established ci MIC cell lines.But the SV40 T antigen-mediated immortalization of the primary MICs is conditional,and that the long-term maintenance of these cells still requires the presence of intestinal stem cell niche factors in the ENR medium;(2)The ci MICs maintained the biological properties of intestinal epithelial stem cells and can form intestinal organoids or Mini-guts in 3D culture conditions.(3)The ci MIC cell lines werenon-tumorigenic,but exhibit high proliferative activity after the introduction of overexpressed oncogenes and develop in vivo intestinal adenomatoid pathological and histological characteristics.