Preliminary Research Of MiR-149 Responding To Folic Acid Deficiency And Affecting One-Carbon Metabolism In Human NCM460 Cell Line

One-carbon metabolism(OCM) is the process of transferring one-carbon from donor compounds to receptors and involves in the process of related biochemical reactions. Folic acid(FA), an oxidated form of folate, is one of core components in OCM and is critical for essential processes including DNA synthesis and methylation.Micro RNAs(mi RNAs) are non-coding small RNAs(-22nt), which can degrade m RNAs or inhibit protein translation by pairing completely or incompletely with m RNAs. We have been predicted that mi R-149 can target MTHFR, a a key gene of OCM, using bioinformatics software. We hypothesized that mi R-149 would response to FA deficiency and affect OCM by targeting and affecting MTHFR m RNA.In this study, the dual-luciferase reporter assay system was used to detect whether the mi R-149 binds directly to the predicted binding sites and to determine whether mi R-149 degrades the MTHFR m RNA or inhibits its translation. Human colorectal normal cells NCM460 were cultured in FA deficient or normal FA concentration culture medium for 28 d. Then RT-q PCR and Poly(A) Tailing RT-q PCR was used to detect the expression of MTHFR and mi R-149. We studied whether mi R-149 would response to FA deficiency and affect OCM by targeting and degrading MTHFR m RNA in NCM460.The results showed that:(1) The data from luciferase analysis showed that mi R-149 inhibited the activity of the reporter vector carrying wild MTHFR 3’UTR(p<0.01), but mutational MTHFR3’UTR(p>0.05) in human embryonic kidney cells HEK293, suggesting that mi R-149 might bind directly to the predicted MTHFR 3’UTR binding sites, and might degrade MTHFR m RNA or inhibit protein translation.(2) Compared with control group, mi R-149 expression was identified as significantly up-regulated(p<0.01) and MTHFR m RNA level was significantly down-regulated(p<0.01) in FA deficiency group, suggesting that mi R-149 might response to FA deficiency and affect OCM by targeting and degrading MTHFRexpression.In summary, these results indicated that mi R-149 responsed to FA deficiency,and affected OCM and the related biological processes by degrading MTHFR expression.