Folic Acid Deficiency Affects The Genome Stability And The Expression Of HMSH2 Regulated By MiR-21-5p In Human Colonic Cells

Folic acid(FA),the most oxidized and stable form of folate,is the key component of one carbon unit metabolism.The exogenous folate are progressively reduced into 5,10-methylenetetrahydrofolate,which is the main methyl donor for deoxyuridine monophosphate(d UMP)and de novo synthesis of deoxythymine monophosphate(d TMP),folic acid deficiency leads to an increase ratio of d UMP /d TMP,resulted in excessive of d UTP incorporating into DNA and increased genomic instability(GIN).Mismatch repair(MMR)system maintains genomic integrity by repairing base mismatches that would occur during DNA replication or recombination.The research fouced on the effect of FA deficiency on genomic stability and expression of h MSH2 by mi R-21-5p in human colonic cells.The aim of the study was to compare the differences of the genomic stability of cells with different physiological and pathological status under the condition of FA deficiency,and to reveal the mechanisms of mi RNA responding FA deficiency and regulating the expression of mismatch repair gene.Accoding to the concentration of FA for maintaining the genome stability in human lymphocytes,we intervened human normal colon epithelial cells CCD-841-Co N and human colorectal adenocarcinoma cell Caco-2 at the FA concentrations of sufficient(2260n M)and deficiency(22.6n M)for 7 days and 21 days.The cytokinesis-block micronucleus cytome assay(CBMN-Cty)was used to evaluate the genomic damage of the tested cells;Poly(A)tailing method was used to analyze the expression of mi R-21-5p under FA deficiency;dual luciferase reporter gene detection system was used to analyze the targetting effect of mi R-21-5p on h MSH2;RT-q PCR and western blotting were used to detect the m RNA expression and protein level of h MSH2 in the tested cells,respectively.The results showed that:(1)CBMN-Cty data revealed that GIN of two tested cells increased with FA deficiency after 21 day culture;the frequency of micronucleus(MN)correlated with chromosome breakage in GIN was significantly increased as well(P<0.01).(2)Dual luciferase reporter assay system analysis confirmed that mi R-21-5p directly targets the 3’UTR of h MSH2 gene.(3)Folic acid deficiency induced up-regulation of the expression of mi R-21-5p and h MSH2 m RNA on the 21 st day in CCD-841-Co N cells(P<0.001).There is no significant change on h MSH2 protein level.(4)Folic acid deficiency induced up-regulation the expression of mi R-21-5p and down-regulation m RNA expression of h MSH2(P<0.01)on the 21 st day in Caco-2 cells.A down-regulation of h MSH2 protein was found,It is suggested that mi R-21-5p may play a different role in the regulating the expression of h MSH2 in different cells.In summary,this study confirms that FA deficiency induces the rise of GIN and mainly in the form of DNA and chromosome breakage in human colorectal cell lines;Under the condition of FA deficiency,the expression of mi R-21-5p was significantly up-regulated in Caco-2 cells,which has also significantly inhibited the m RNA and protein expression of h MSH2,which blocked DNA repair ability;However,in CCD-841-Co N cells,mi R-21-5p and h MSH2 m RNA expression was significantly up-regulated,but not in protein levels.It suggested that the mi RNA-21-5p responds to the deficiency of FA and differentially regulates h MSH2 in different physiological and pathological cells.