Study On Relationships Of One Carbon Metabolisim Bvitamins, MGMT Genemethylation And Esophageal Cancer And Precancerouslesions

Objective:To explore the risk factors of esophageal squamous oell carcinoma (ESCC) and esophageal precancerous lesions (EPL) in Huai’an District, Jiangsu Province, where is a high-incidence area of esophageal cancer, and to understand the role and interactions of MGMT gene promoter region methylation, several one-carbon metabolism related B vitamins (folate, VB2, VB6, VB12) serum concentrations in the carcinogenesis of esophageal precancerous lesions (EPL) and esophageal squamous cell carcinoma (ESCC). Human normal esophageal epithelial cell cultural techniques was used in this paper to observe the effect of AFB1 on human normal esophageal epithelial cell proliferation, cell cycle and apoptosis, MGMTgene promoter region methylation. Meanwhile, the relationship between one-carbon metabolism related B vitamins and esophageal cancer was analyzed and verified intervention in vitro, so that providing an effective moleculer biomarkers for early diagnosis of ESCC and establishing a scientific basis for establishing measures for primary prevention of esophageal cancer.Methods (1) 220 patients with ESCC 220 patients with EPL and 220 age-, and sex-matched healthy controls were recruited in a high esophageal cancer incidence region, Huai’an district, Huai’an City, China, collecting blood samples and extracting DNA from blood samples. Methylation Specific PCR was used to measure MGMT gene promoter region methylation. (2) The enzyme-linked immunosorbent assay was used to detect the serum folicacid, VB2, VB6, VB12 levels and then the differences between esophageal cancer, precancerous lesions cases and control group were compared. Then the serum vitamins which were significantly different among three groups were divided into four levels according to control group’s vitamin level.Gene-nutrient interactions were evaluated using multinomial logistic regression models by combining the MGMT gene promoter region methylation and serum vitamins quartiles.(3) Methyl Thiazolyl Tetrazolium and MSP was used to identified the effect of AFB1 on human normal esophageal epithelial cell proliferation, cell cycle and apoptosis and MGMT gene promoter region methylation.(4) Methyl Thiazolyl Tetrazolium and gene chip technique was used to identified the effect of folate, VB2, VB6 and VB12 on human normal esophageal epithelial cell proliferation, cell cycle and apoptosis and MGMT gene promoter region methylation which were treating with AFB1.Results;(1)The rate of MGMT gene methylation in three groups were 37.3%(82/220), 19.1%(41/220) and 3.8%(8/220) separately. There was a significant difference in the DNA methylation frequency of MGMTgene among the ESCC, EPL and control groups (P<0.05).(2) The serum folate and VB6 levels may further modify the association of MGMT gene methylation with the risk of ESCC and EPL however the serum VB2 and VB12 leves may not.(P <0.05). Campared with the minimum quartile of serum folate, the second quartile of serum folate may further increase the risk of ESCC and/or EPL when MGMT gene wasn’t methylated(P<0.05). The risk of ESCC and/or EPL may decreased when the serum folate level was high (>26.92) whether MGMT gene methylated or not(P<0.05). MGMT gene mathylation may increased the risk of ESCC and/or EPL when the serum folate level in the lowest quartile(P<0.05). Campared with the minimum quartile of serum folate, the serum folate level in highest quartile may further increase the risk of ESCC and/or EPL. whether MGMT gene methylated or not(P<0.05). (3)The results showed that the proliferation of human normal esophageal epithelial cells was decreased when cells were treated with different concentrations of 0.2μg/mL,0.4μg/mL,0.8p.g/mL,1.6μg/mL,2μg/mL AFB1 for 24h、 48h and 72h respectively. When cells were treated with 0.8μg/mL、1.6μg/mL、2μg/mL AFB1 for 48h separately, G0/G1 phase cell percentage were significantly lower than that of Oμmol/L AFB1 group (p<0.05). Sphase cells proportios and apoptosis ratio were higher than that of Oμmol/L AFB1 group when the AFB1 concentrations wereμg/mL,0.8μg/mL,16μg/mL and 2μg/mL (p< 0.05). When cells were treated with 1.6μg/mL and 2μg/mL AFB1 for 48h, G2/M phase cell percentage was significantly higher than that of Oμmol/L AFB1 group (p< 0.05). The MGMT gene was methylated when the the AFB1 concentrations was 2μg/mL(4) Human normal esophageal epithelial cells with 2μg/mL AFB1 while adding 5μmol/L folate and other B vitamins were treated for 48h could promote cell proliferation, and the G2/M phase, s phase cells, apoptosis ratio were lower than control group.Meanwhile the methylation rate of CpG14-15 on MGMT gene promoter was higher than control group when added 5μmol/L folate and 5μmol/L VB6(p<0.05)Conclusion:The occurrence of esophageal cancer and precancerous lesions could be associated with MGMT gene promoter region DNA methylation. And there was some interactions between sever B vitamins and genetic factors. AFBi can inhibit human normal esophageal epithelial cell proliferation and promot apoptosis, promote cells from G0/G1 phase into S phase, block at S and G2/M phase, it also can make mgmt gene being mathylated. Intervention in vitro showed B vitamins could promote AFB1 cell proliferation, improve AFB1 in Sphase block, promote cells enter G2/M phase, block AFB1 on human normal esophageal epithelial cells apoptosis promotion and block AFB1 inducing MGMT gene methylation.