The Relationship Between The Risk Of Esophageal Cancer And One-Carbon Metabolism Related B Vitamins And The Influence Of These Vitamins On The Cell Proliferation Of Microcystins-LR

Objective:1.A meta-analysis was conducted to assess the relationship between both the levels of folic acid and other one-carbon metabolism related B vitamins(vitamin B₂,vitamin B₆,vitamin B₁₂)and the risk of esophageal cancer.2.To investigate the effect of microcystin-LR on the activity of normal human esophageal epithelial cells and its influence on inducing cell apoptosis.3.To investigate the effects of folic acid and vitamin B₆ on human normal esophageal epithelial cells exposed to microcystin-LR and observe their effects on cell proliferation,cell cycle,cell apoptosis,the mRNA levels and protein levels of P53 and P16.Investigate the inhibitory effect of folic acid and vitamin B₆ on cell proliferation induced by microcystin-LR,and provide theoretical basis for the further study on the effects of folic acid and vitamin B₆ on the prevention and control of microcystin-LR to influence human health.To provide scientific basis for carrying out nutritional intervention in high incidence areas of esophageal cancer in China.Methods and Results:1.According to the guide of Meta-analysis for epidemiological studies,a detailed inclusion and exclusion criteria was established to identify eligible studies published until May 20,2018,and relevant data were extracted from the literature to analyze.This meta-analysis was conducted to summarize the pooled odds ratio?ORs?as the statistic of the impact of each exposure factor on the risk of esophageal cancer,Stata 11.0 software was used to test the heterogeneity of each study and select the appropriate model to calculate the combined OR value as well as its 95%confidence interval?95%Confidence Interval,95%CI?.Pre-specified subgroup analyses were conducted to explore potential heterogeneity.Begg's test and Egger's test were chosen to evaluate the publication bias.A total of 25 studies were included in this Meta-analysis,the pooled OR and 95%CI for folic acid,vitamin B₂,vitamin B₆ and vitamin B₁₂ associated with the risk of esophageal cancer were 0.63?0.54-0.74?,1.03?0.88-1.20?,0.59?0.48-0.72?and 1.26?1.01-1.59?,respectively.The effect amount I² of the heterogeneity test were 53.6%,37.6%,64.6%,71.8%,respectively.2.Human normal esophageal epithelial cells were treated with low dose of microcystin-LR?0.01,0.001,0.0001,0.00001,0.000001?g/mL?for 24 h,CCK8 cell proliferation toxicity test kit?Cell Counting Kit-8,CCK8?was used to detect cell viability,then an appropriate concentration of microcystin-LR was selected to establish the dosage of the cell injury model and mimic the process of cancer cell transformation.Human normal esophageal epithelial cells were treated with high doses of microcystin-LR?1,3.75,7.5,15,30,and 50?g/mL?for 24 h,and cell viability was measured by CCK8.The 50%inhibitory concentration(IC₅₀)was calculated to be 24?g/mL,so 6,12,24?g/mL(1/4 IC₅₀,1/2 IC₅₀,IC₅₀)microcystin-LR were used as the experimental concentration of inducing cell apoptosis.Human normal esophageal epithelial cells were treated with 6,12 and 24?g/mL microcystin-LR for 24 hours.Apoptosis was detected by Annexin V-FITC/PI double staining;PCR was used to detect the mRNA expression levels of Caspase-3 and Caspase-9.Western blot was performed to detect the protein expression levels of Caspase-3 and Caspase-9.Compared with the control group,low dose of microcystin-LR can increase cell viability,among which 0.0001 to 0.00001?g/mL of microcystin-LR has the most significant effect on the improvement of human normal esophageal epithelial cell viability.Therefore,0.00005?g/mL of microcystin-LR was selected as the dose of the cell injury model.After treating human normal esophageal epithelial cells with different concentrations of microcystins-LR for 24 h,3.75-50?g/mL of microcystin-LR can make normal esophageal epithelium more stable than the control group.Cell viability decreased and the difference was statistically significant.Compared with the control group,6,12 and 24?g/mL microcystins-LR could induce cell apoptosis and increase the apoptosis rate,also can increase the mRNA and protein expression of Caspase-3 and Caspase-9.3.Human normal esophageal epithelial cells were treated with 0.00005?g/mL microcystin-LR,folic acid and vitamin B₆ were added simultaneously,and CCK8 was used to detect cell viability.Cell apoptosis was detected by Annexin V-FITC/PI double staining;mRNA expression of P16 and P53were detected by PCR;protein expression levels of P16 and P53 were detected by WB.Compared with the microcystin-LR exposure model group,the addition of 200?g/mL folic acid and vitamin B₆ can inhibit normal human esophageal epithelial cell proliferation.Compared with the control group,cell apoptotic rate was decreased,the proportion of cells in S phase increased,and the expression of P16 and P53mRNA and protein were down-regulated after 0.00005?g/mL microcystin-LR treatment for24 h.After folic acid and vitamin B₆ intervention,compared with the microcystin-LR exposure model group,the cell apoptosis rate increased,the proportion of S phase cells decreased,and the expression of P16 and P53 mRNA and protein was increased.Conclusion:1.This meta-analysis revealed that dietary folic acid intake and vitamin B₆ levels were negatively correlated with the risk of esophageal cancer;the levels of vitamin B₁₂ were positively correlated with the risk of esophageal cancer;the association between serum folic acid levels,serum vitamin B₆ levels and vitamin B₂ levels and the risk of esophageal cancer was insignificant.2.Low concentration of microcystins-LR can increase normal human esophageal epithelial cell viability and high concentrations of microcystin-LR can induce normal human esophageal epithelial cell apoptosis and may be associated with apoptosis-related signaling molecules Caspase-3 and Caspase-9.3.Low doses of microcystin-LR can promote normal human esophageal epithelial cell proliferation and inhibit cell apoptosis;folic acid and vitamin B₆ intervention inhibits normal human esophageal epithelial cells proliferation exposed to microcystin-LR.