The Role Of Folic Acid On MNNG-induced DNA Damage And Related Signaling Transduction Pathway In DNMT1 High Expression Kazakh Esophageal Epithelial Cells

Objective:Promote the occurrence and development of the high expression of DNMT1 in tumor transformation and DNA methylation patterns in the early has established a foundation in the human immortalized esophageal epithelial cell line on the construction of the high expression of DNMT1 in esophageal epithelial cell line by used TALE technology,Analyze the role of folic acid in MNNG-induced DNA damage of Kazak’s esophageal epithelial cells that express high levels of DNMT1 and the related signaling pathway regulating mechanism,and explore the intervention by folic acid in cell damage,to provide theoretical basis for the prevention and treatment of esophageal cancer.Methods:Kazak’s esophageal epithelial cells and Kazak’s esophageal epithelial cells expressing high levels of DNMT1 were assigned respectively to three groups and MNNG were exposed and were interventiond with low concentration of folic acid,folic acid medium concentration and high concentration of folic acid,The growth of the grouped cells was observed with an inverted fluorescence microscope.Single cell gel electrophoresis assay(comet assay)was performed to examine the DNA damages,and RT-PCR and Western blot were used to determine the mRNA and protein expression levels of PI3K-AKT signaling pathway,PP2 A,PTEN and AKT in the grouped cells,respectively.Results:(1)The damages to cell morphology in different exposure time got worse as the exposure time increased in both types of cells,and the DNMT1-high cells were affected more severely by MNNG than normal esophageal epithelial cells.However,cells from the high-dose folic acid group grew better,had lower death rate,were better organized and showed better morphology nearer to that of the controls,than their counterparts in the low and median-dose folic acid groups in the same class at the same period of exposure.(2)The DNA damage in different exposure time got worse as the exposure time increased in both types of cells,and wasmore severe in DNMT1-high esophageal epithelial cells than in normal esophageal epithelial cells.as a long tail in the early and middle and late period of exposure,later than normal cells group,the difference was statistically significant(tearly stage=2.043,P=0.004;tmediumstage=2.217,P=0.030;tlate stage=2.418,P=0.016),the high expression of DNMT1 cells Olive tail length The difference was statistically significant in the early and middle and late stages than in the normal cell group(tearlystage=2.471,P=0.020;tmediumstage=2.412,P=0.016;tlate stage=2.047,P=0.004).Cells from the high-dose folic acid group had less severe damage,and had lower percentage of DNA in tail,tail length and Oliver tail moment than their counterparts in the low and median-dose folic acid groups in the same class at the same period of exposure(P<0.05).(3)The mRNA and protein expression levels of PP2 A and PTEN genes lowered as the exposure time increased in both types of cells,and were lower in DNMT1-high esophageal epithelial cells than in normal esophageal epithelial cells from the same group at the same period of exposure.The mRNA and protein expression levels of AKT genes increased as the exposure time increased in both types of cells,and were higher in DNMT1-high esophageal epithelial cells than in normal esophageal epithelial cells from the same group at the same period of exposure(P < 0.05).Cells from the high-dose folic acid group had higher PP2 A and PTEN mRNA and protein expression levels and lower AKT mRNA and protein expression levels than their counterparts in the low and median-dose folic acid group in the same class at the same period of exposure(P< 0.05).Conclusion: Kazak’s esophageal epithelial cells that express high levels of DNMT1 are more susceptible to MNNG than Kazak’s normal esophageal epithelial cells,and cell damages get worse as the exposure time increases.showed morphological changes,DNA damage,and lead to the down-regulation of PP2 A gene and PTEN gene mRNA and protein expression in PI3K-AKT pathway,AKT gene mRNA and protein expression;However,high-concentration folic acid can reduce the damages to the cells caused by MNNG,protect the normal expression of PI3K-AKT pathway in PP2 A,PTEN,AKT gene,and maintaining sufficient folic acid intake plays certain protective role in the development and progression of esophageal cancer.