γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.

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Global Leukocyte DNA Methylation is Similar in African American and Caucasian Women Under Conditions of Controlled Folate Intake

DNA methylation is an epigenetic feature that may modify disease risk, and can be influenced by folate status as well as by methylenetetrahydrofolate reductase (MTHFR) C677T genotype. The aim of this study was to investigate the influence of ethnicity/race on global leukocyte DNA methylation under conditions of controlled folate intake. Caucasian (n=14) and African American (n-14) women (18-45y) possessing the MTHFR 677CC genotype consumed a folate restricted diet (135 μg/d as dietary folate equivalents, DFE) for 7 week followed by folate treatment with 400 or 800 μg DFE/d for 7 week. Global leukocyte DNA methylation was assessed via the cytosine extension assay at baseline (wk 0), after folate restriction (wk 7) and after folate treatment (wk 14). Ethnicity/race was not a determinant of global leukocyte DNA methylation. No differences (P>0.05) were detected in DNA methylation between African American and Caucasian women at baseline or any other study time point. In addition, folate intake did not modify global leukocyte DNA methylation. These data suggest that global leukocyte DNA methylation does not differ between Caucasian and African American women and that short-term folate restriction is not sufficient to modify methylation content in young women with the MTHFR 677CC genotype.     

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994–1995), peri- (1996–1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.     

No effect of folic acid supplementation on global DNA methylation in men and women with moderately elevated homocysteine

A global loss of cytosine methylation in DNA has been implicated in a wide range of diseases. There is growing evidence that modifications in DNA methylation can be brought about by altering the intake of methyl donors such as folate. We examined whether long-term daily supplementation with 0.8 mg of folic acid would increase global DNA methylation compared with placebo in individuals with elevated plasma homocysteine. We also investigated if these effects were modified by MTHFR C677T genotype. Two hundred sixteen participants out of 818 subjects who had participated in a randomized double-blind placebo-controlled trial were selected, pre-stratified on MTHFR C677T genotype and matched on age and smoking status. They were allocated to receive either folic acid (0.8 mg/d; n = 105) or placebo treatment (n = 111) for three years. Peripheral blood leukocyte DNA methylation and serum and erythrocyte folate were assessed. Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry and expressed as a percentage of 5-methylcytosines versus the total number of cytosine. There was no difference in global DNA methylation between those randomized to folic acid and those in the placebo group (difference = 0.008, 95%CI = -0.05,0.07, P = 0.79). There was also no difference between treatment groups when we stratified for MTHFR C677T genotype (CC, n = 76; CT, n = 70; TT, n = 70), baseline erythrocyte folate status or baseline DNA methylation levels. In moderately hyperhomocysteinemic men and women, long-term folic acid supplementation does not increase global DNA methylation in peripheral blood leukocytes.ClinicalTrials.gov NCT00110604.     

Folate depletion during pregnancy and lactation reduces genomic DNA methylation in murine adult offspring

The developmental origins of adult health and disease (DOHaD) hypothesis that argues for a causal relationship between under-nutrition during early life and increased risk for a range of diseases in adulthood is gaining epidemiological support. One potential mechanism mediating these effects is the modulation of epigenetic markings, specifically DNA methylation. Since folate is an important methyl donor, alterations in supply of this micronutrient may influence the availability of methyl groups for DNA methylation. We hypothesised that low folate supply in utero and post-weaning would alter the DNA methylation profile of offspring. In two separate 2 × 2 factorial designed experiments, female C57Bl6/J mice were fed low- or control/high-folate diets during mating, and through pregnancy and lactation. Offspring were weaned on to either low- or control/high-folate diets, resulting in 4 treatment groups/experiment. Genomic DNA methylation was measured in the small intestine (SI) of 100-day-old offspring. In both experiments, SI genomic DNA from offspring of low-folate-fed dams was significantly hypomethylated compared with the corresponding control/high folate group (P = 0.009/P = 0.006, respectively). Post-weaning folate supply did not affect SI genomic DNA methylation significantly. These observations demonstrate that early life folate depletion affects epigenetic markings, that this effect is not modulated by post-weaning folate supply and that altered epigenetic marks persist into adulthood.     

Methylenetetrahydrofolate reductase 677C-->T polymorphism affects DNA methylation in response to controlled folate intake in young women

DNA methylation is critical for normal genomic structure and function and is dependent on adequate folate status. A polymorphism (677C-->T) in a key folate enzyme, methylenetetrahydrofolate reductase (MTHFR), may impair DNA methylation when folate intake is inadequate and may increase the risk of reproductive abnormalities. The present study was designed to evaluate the effect of the MTHFR 677C-->T polymorphism on changes in global DNA methylation in young women consuming a low folate diet followed by repletion with the current Recommended Dietary Allowance (RDA). Women (age 20-30 years) with the TT (variant; n = 19) or CC (n = 22) genotype for the MTHFR 677C-->T polymorphism participated in a folate depletion-repletion study (7 weeks, 115 microg DFE/day; 7 weeks, 400 microg DFE/day). DNA methylation was measured at baseline, week 7, and week 14 using a [3H]methyl acceptance assay and a novel liquid chromatography tandem mass spectrometry assay of the DNA bases methylcytosine and cytosine. [3H]Methyl group acceptance tended to increase (P = 0.08) during depletion in all subjects, indicative of a decrease in global DNA methylation. During repletion, the raw change and the percent change in the methylcytosine/total cytosine ratio increased (P = 0.03 and P = 0.04, respectively) only in the subjects with the TT genotype. Moderate folate depletion in young women may cause a decrease in overall DNA methylation. The response to folate repletion suggests that following folate depletion women with the MTHFR 677 TT genotype have a greater increase in DNA methylation with folate repletion than women with the CC genotype.     

Effect of substrate competition in kinetic models of metabolic networks

Substrate competition can be found in many types of biological processes, ranging from gene expression to signal transduction and metabolic pathways. Although several experimental and in silico studies have shown the impact of substrate competition on these processes, it is still often neglected, especially in modelling approaches. Using toy models that exemplify different metabolic pathway scenarios, we show that substrate competition can influence the dynamics and the steady state concentrations of a metabolic pathway. We have additionally derived rate laws for substrate competition in reversible reactions and summarise existing rate laws for substrate competition in irreversible reactions.     

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

Inhibition of DNA demethylation enhances plant tolerance to cadmium toxicity by improving iron nutrition

Although the alteration of DNA methylation due to abiotic stresses, such as exposure to the toxic metal cadmium (Cd), has been often observed in plants, little is known about whether such epigenetic changes are linked to the ability of plants to adapt to stress. Herein, we report a close linkage between DNA methylation and the adaptational responses in Arabidopsis plants under Cd stress. Exposure to Cd significantly inhibited the expression of three DNA demethylase genes ROS1/DML2/DML3 (RDD) and elevated DNA methylation at the genome-wide level in Col-0 roots. Furthermore, the profile of DNA methylation in Cd-exposed Col-0 roots was similar to that in the roots of rdd triple mutants, which lack RDD, indicating that Cd-induced DNA methylation is associated with the inhibition of RDD. Interestingly, the elevation in DNA methylation in rdd conferred a higher tolerance against Cd stress and improved cellular Fe nutrition in the root tissues. In addition, lowering the Fe supply abolished improved Cd tolerance due to the lack of RDD in rdd. Together, these data suggest that the inhibition of RDD-mediated DNA demethylation in the roots by Cd would in turn enhance plant tolerance to Cd stress by improving Fe nutrition through a feedback mechanism.     

Genome‐wide variation in DNA methylation is associated with stress resilience and plumage brightness in a wild bird

Individuals often differ in their ability to cope with challenging environmental and social conditions. Evidence from model systems suggests that patterns of DNA methylation are associated with variation in coping ability. These associations could arise directly if methylation plays a role in controlling the physiological response to stressors by, among other things, regulating the release of glucocorticoids in response to challenges. Alternatively, the association could arise indirectly if methylation and resilience have a common cause, such as early‐life conditions. In either case, methylation might act as a biomarker for coping ability. At present, however, relatively little is known about whether variation in methylation is associated with organismal performance and resilience under natural conditions. We studied genome‐wide patterns of DNA methylation in free‐living female tree swallows (Tachycineta bicolor) using methylated DNA immunoprecipitation (MeDIP) and a tree swallow genome that was assembled for this study. We identified areas of the genome that were differentially methylated with respect to social signal expression (breast brightness) and physiological traits (ability to terminate the glucocorticoid stress response through negative feedback). We also asked whether methylation predicted resilience to a subsequent experimentally imposed challenge. Individuals with brighter breast plumage and higher stress resilience had lower methylation at differentially methylated regions across the genome. Thus, widespread differences in methylation predicted both social signal expression and the response to future challenges under natural conditions. These results have implications for predicting individual differences in resilience, and for understanding the mechanistic basis of resilience and its environmental and social mediators.     

DNA methylation,an epigenetic mechanism connecting folate to healthy embryonic development and aging

Experimental studies demonstrated that maternal exposure to certain environmental and dietary factors during early embryonic development can influence the phenotype of offspring as well as the risk of disease development at the later life. DNA methylation, an epigenetic phenomenon, has been suggested as a mechanism by which maternal nutrients affect the phenotype of their offspring in both honeybee and agouti mouse models. Phenotypic changes through DNA methylation can be linked to folate metabolism by the knowledge that folate, a coenzyme of one-carbon metabolism, is directly involved in methyl group transfer for DNA methylation. During the fetal period, organ-specific DNA methylation patterns are established through epigenetic reprogramming. However, established DNA methylation patterns are not immutable and can be modified during our lifetime by the environment. Aberrant changes in DNA methylation with diet may lead to the development of age-associated diseases including cancer. It is also known that the aging process by itself is accompanied by alterations in DNA methylation. Diminished activity of DNA methyltransferases (Dnmts) can be a potential mechanism for the decreased genomic DNA methylation during aging, along with reduced folate intake and altered folate metabolism. Progressive hypermethylation in promoter regions of certain genes is observed throughout aging, and repression of tumor suppressors induced by this epigenetic mechanism appears to be associated with cancer development. In this review, we address the effect of folate on early development and aging through an epigenetic mechanism, DNA methylation.     

The effect of GNAQ methylation on GnRH secretion in sheep hypothalamic neurons

Kazakh sheep are seasonal estrous animals, and gonadotropin-releasing hormone (GnRH) is the key to fertility regulation. The nutritional level has a certain regulatory effect on estrous, and vitamin B folate plays a role in DNA methylation, directly participating in the process. The goal of this study was to determine whether folate is involved in GnAQ methylation and its effect on GnRH secretion. The hypothalamic neurons of Kazakh fetal sheep were treated with folate at concentrations of 0 mg/mL, 4 mg/mL, 40 mg/mL, and 80 mg/mL. GnAQ promoter methylation, DNMT1, GnAQ expression, and GnRH secretion following treatment with different concentrations of folate were analyzed. One CpG site was methylated in the GNAQ promoter with 40 mg/mL folic acid, and no CpG methylation was found in the other groups. GnAQ expression was related to folate concentration and showed a trend of increasing first and then decreasing. The GnRH expression level in the 40 mg/mL folate group was significantly higher than in the other three groups ( P < .05). These results demonstrate that the appropriate folate concentration promoted GANQ promoter methylation, which in turn affected GnRH secretion.     

Methylation of the PTPRO gene in human hepatocellular carcinoma and identification of VCP as its substrate

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type‐O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate‐trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate‐trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild‐type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy. J. Cell. Biochem. 114: 1810–1818, 2013. © 2013 Wiley Periodicals, Inc.     

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994–1995), peri- (1996–1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.     

Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.     

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.     

LINE-1 DNA methylation is inversely correlated with cord plasma homocysteine in man: A preliminary study

Folic acid supplementation during pregnancy has known beneficial effects. It reduces risk of neural tube defects and low birth weight. Folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. However, most data on the effects of folate on the epigenome is derived from animal or in vitro models. We examined the relationship between cord blood methylation and maternal folic acid intake, cord blood folate and homocysteine using data from 24 pregnant women. Genome-wide methylation was determined by the level of methylation of LINE-1 repeats using Pyrosequencing. We show that cord plasma homocysteine (p = 0.001, r = -0.688), but not serum folate or maternal folic acid intake, is inverse correlated with LINE-1 methylation. This remained significant after correction for potential confounders (p = 0.004). These data indicate that levels of folate-associated intermediates in cord blood during late pregnancy have significant consequences for the fetal epigenome.     

Erythrocyte folate concentrations,CpG methylation at genomically imprinted domains,and birth weight in a multiethnic newborn cohort

Epigenetic mechanisms are proposed to link maternal concentrations of methyl group donor nutrients with the risk of low birth weight. However, empirical data are lacking. We have examined the association between maternal folate and birth weight and assessed the mediating role of DNA methylation at nine differentially methylated regions (DMRs) of genomically imprinted genes in these associations. Compared with newborns of women with folate levels in the lowest quartile, birth weight was higher in newborns of mothers in the second (β = 143.2, se = 63.2, P = 0.02), third (β = 117.3, se = 64.0, P = 0.07), and fourth (β = 133.9, se = 65.2, P = 0.04) quartiles, consistent with a threshold effect. This pattern of association did not vary by race/ethnicity but was more apparent in newborns of non-obese women. DNA methylation at the PLAGL1, SGCE, DLK1/MEG3 and IGF2/H19 DMRs was associated with maternal folate levels and also birth weight, suggestive of threshold effects. MEG3 DMR methylation mediated the association between maternal folate levels and birth weight (P =0.06). While the small sample size and partial scope of examined DMRs limit our conclusions, our data suggest that, with respect to birth weight, no additional benefits may be derived from increased maternal folate concentrations, especially in non-obese women. These data also support epigenetic plasticity as a key mechanistic response to folate availability during early fetal development.     

Folate deficiency disturbs hsa‐let‐7 g level through methylation regulation in neural tube defects

Folic acid deficiency during pregnancy is believed to be a high‐risk factor for neural tube defects (NTDs). Disturbed epigenetic modifications, including miRNA regulation, have been linked to the pathogenesis of NTDs in those with folate deficiency. However, the mechanism by which folic acid‐regulated miRNA influences this pathogenesis remains unclear. It is believed that DNA methylation is associated with dysregulated miRNA expression. To clarify this issue, here we measured the methylation changes of 22 miRNAs in 57 human NTD cases to explore whether such changes are involved in miRNA regulation in NTD cases through folate metabolism. In total, eight of the 22 miRNAs tested reduced their methylation modifications in NTD cases, which provide direct evidence of the roles of interactions between DNA methylation and miRNA level in these defects. Among the findings, there was a significant association between folic acid concentration and hsa‐let‐7 g methylation level in NTD cases. Hypomethylation of hsa‐let‐7 g increased its own expression level in both NTD cases and cell models, which indicated that hsa‐let‐7 g methylation directly regulates its own expression. Overexpression of hsa‐let‐7 g, along with its target genes, disturbed the migration and proliferation of SK‐N‐SH cells, implying that hsa‐let‐7 g plays important roles in the prevention of NTDs by folic acid. In summary, our data suggest a relationship between aberrant methylation of hsa‐let‐7 g and disturbed folate metabolism in NTDs, implying that improvements in nutrition during early pregnancy may prevent such defects, possibly via the donation of methyl groups for miRNAs.