The Effects Of Folate With Different Reduced Status On H TERT And HMLH1 Transcription In Human A375 And BJ Cell Lines

Telomere is a region of 5-15 kb "5-TTAGGG-3" short tandem repetitive sequences with special binding proteins, located on the chromosome ends to protect the ends of chromosome. Telomerase is a specialized reverse transcriptase and its key subunit hTERT catalyzes the telomere elongation. The telomeric DNA could be shorten for 30-150 bp with each cell division in most human somatic cells since lack of telomerase activity, finally toward cellular senescence. There is positive correlation between the expression of hTERT,and the activity of telomerase, as well as telomere length.DNA mismatch repair is an important system in eukaryocytes, which functions in post-replicative repairing of erroneous insertion, deletion, and mis-incorporation of bases. The hMLH1 protein is a core component of mismatch repair system, which participates in the correcting mismatched base during DNA replication and maintains genomic stability.Folate is a water-soluble B vitamins, it is the key micromutrent in one carbon unit metabolism. Folate deficiency could block the synthesis of dTMP and results in dUMP misincorporation in DNA eventually leading to DNA mismatch and chromosome breakage.The research focused on the relationship between folate deficiency and the transcription of hMLH1 and hTERT in human telomerase positive cell line A375 and negative cell line BJ. 22.66, 226.60 and 2266.00 nmol/L of folic acid or 5-methyltetrahydrofolate modified RPMI-1640 were set for the cell intervention for 21 d. Total RNA was isolated on the day of 7, 14 and 21 after the intervention. The mRNA levels of hMLH1 and hTERT were determined by relative real-time quantitative PCR.The results showed that:(1) Compare to the control, each concentration significantly decreased hTERT mRNA level in A375 cells(p<0.05), Both FA and 5-MeTHF significantly decreased hTERT mRNA level in A375 under the concentration of 22.66 nmol/L with the extention of intervention time than control and middle/high concentration groups(226.60 and 2266.00 nmol/L)(p<0.05), there are no significant difference with follow two high concentration groups, suggesting that above 226.60 nmol/L folate(FA and 5-MeTHF) concentration can maintain A375 hTERT transcription. Under the same intervention condition, BJ hTERT mRNA levels were not affected by the folate intervention, still silenced.(2) Both FA and 5-MeTHF significantly decreased hMLH1 mRNA levels in A375 under the concentration of 22.66 nmol/L with the extention of process time than control and middle/high concentration groups(226.60 and 2266.00 nmol/L)(p<0.05), However constant expression at the concentration of 226.60 and 2266.00 nmol/L and lower than the control, suggesting that folate deficiency could reduced DNA damage repair ability at this kind of cell, appropriate folate supply could recovery the DNA damage repair ability of cells. On the contrary, BJ hMLH1 mRNA levels were not significantly different to the control under the low concentration FA(22.66 nmol/L), but in the middle and high concentration group(226.60 and 2266.00 nmol/L), hMLH1 mRNA transcription levels were significantly increased compared to the control with the intervention process(p<0.05); To this two test cell line, 226.60 nmol/L and above of FA and 5-MeTHF could contribute to DNA mismatch repair ability recover even improved.