Study On The Mechanism Of Stemness And Pluripotency Maintenance Of MESCs Induced By Folic Acid Supplementation

Birth defects are an important global public health issue that has a wide range of impacts.In some countries birth defects are also a major cause of early spontaneous abortion,termination of pregnancy,death of infants,and disability.According to reports,nearly 70% of birth defects can be prevented or cured with good care.As an important microelement,folic acid has been recognized for its role in preventing birth defects.For the above reasons,many countries have begun to implement mandatory folic acid supplementation of the entire population as a public health policy to prevent birth defects.However,the exact relationship between folic acid and development has not yet been clarified.In particular,the differences in folic acid uptake ability of different individuals are geater,and the harm caused by folic acid supplementation to birth defects or the damage caused by folic acid excess needs to be further clarified.In this study,we established an animal model of different folic acid feeds to the parental mouse.We found that the expression of the stemness marker molecule in the liver of the progeny mouse increased as the level of folic acid in the body increased.The effects of maternal folate levels on the growth and development of offspring mice were clarified.We used E14 mouse embryonic stem cells(mESCs)as subjects to examine the biological characteristics,stemness,and pluripotency of mESCs cultured at different folic acid concentrations for 6 months.The subcutaneous teratoma model of mESCs injected into nude mice was successfully established.Through the above experiments,we found that the supplementation of folic acid can enhance its in vitro proliferation,expression of colony formation and stemness-related markers,and maintain its pluripotency well in vivo compared to conventionally cultured mESCs.After clarifying the relative effects of folic acid on embryonic stem cells,we further explored the mechanism by which folic acid supplementation promotes the proliferation of embryonic stem cells.Differentially expressed genes from cultured cells were analyzed by transcriptome sequencing technology to finally screen and confirm a maternally imprinted gene imprinted long non-coding RNA(lncRNA)Meg3 expressed in mESCs at high folate levels(maternally expressed gene 3).Bisulfite Genomic Sequence have shown that this upregulation of Meg3 is caused by hypomethylation of the promoter,and that it can restore folic acid supplementation by regulating the expression of Meg3 in the cell.Through further mechanism studies,we successfully confirmed the interaction between Meg3 and LYAR proteins(Cell growthregulating nucleolar protein).The results show that Meg3 stabilizes LYAR by preventing the ubiquitination and degradation of LYAR,and the effect of Meg3 on the stemness of mESC depends on the stability of LYAR.In conclusion,we conclude that maternal high folate intake may have a series of effects on the offspring’s growth and development,illustrating the correlation between long-term high folic acid intake and offspring birth defects and maternal imprinting lncRNA Meg3 involved in long-term folic acid intake High folic acid levels can cause hypomethylation in the Meg3 promoter region,induce upregulation of Meg3 expression,thereby maintaining the stability of LYAR,and maintaining the stem cell differentiation and pluripotency of mESCs.Our results provide a theoretical basis for the dialectical role of folic acid supplementation in development and we look forward to our research findings that can help guide clinical folate supplementation.