| Mammalian spermatogenesis is a process containing,proliferation and differentiation of spermatogonia,meiosis and spermatids transformation.This process is elaborately regulated by the epigenetic regulators including transcription factor,histone modification and noncoding RNA.Current research showed that N6-methyladenosine(m6A)is the most prevalent internal modification in eukaryotic mRNA,which has been identified to regulate gene expression through mediating m RNA splicing,decay and translation.In addition,m6 A modification is involved in regulating spermatogenesis.FTO is the first discovered m6 A demethylase,which could regulate the differentiation of adipocytes and the proliferation of cancer cells by altering the level of m6 A modification.MA2,as the ethyl ester form of meclofenamic acid(MA),can effectively bind to the active site of FTO and inhibit the demethylation activity of FTO.In order to investigate the role of m6 A modification on the proliferation of spermatogonia,GC-1 spg cells were selected as the model in this study.By using CCK-8,western blot,EdU,flow cytometry,qRT-PCR,m6 A dot blot to study the functions of m6 A on spermatogonial proliferation.Constructed dual fluorescence reporting system,and further confirmed the function of m6 A reporting plasmid.The results showed as follows:1.MA2 treatment did not change the expression level of FTO,but increased markedly the m6 A modification on mRNA molecules.Moreover,m6 A modifications exhibited a negative correlation with MA2 concentration.2.MA2 treatment resulted in a decrease in cell viability,and remarkably increased multinucleate cells.However,MA2 treatment did not alter the proportion of TUNEL positive cells and the abundance of Cleaved PARP,Cleaved Caspase 3,Bax and Bcl2.3.MA2 treatment reduced significantly the proportion of EdU positive cells and the expression of PCNA and Ki67.In addition,the FTO inhibitor MA2 treatment induced the G1/S arrest of spermatogonia.4.The cell cycle regulatory genes with m6 A modification were obtained from online m6 A database(http://mirlab.sysu.edu.cn/rmbase/).qRT-PCR assay confirmed that the expression of CDK1,CDK2,CDK7,CDK9 and CDK12 was significantly downregulated by MA2 treatment,while CDK8 was up-regulated by 67.7%.5.The CREBBP-m6 A reporter system was constructed from the 3’ UTR region of CREBBP gene containing three m6 A modified sites.The fluorescence intensity of the red fluorescent of mCherry was negatively correlated with the level of m6 A modification.The fluorescent reporter plasmid of CDK2 3’ UTR m6 A modified sites was constructed.The expression of its upstream transcript was regulated by m6 A modification in CDK2 3’ UTR region.In summary,inhibition of FTO by MA2 in GC-1 spg cells increased the level of m6 A and regulated the expression of cell cycle related genes,which further induced arrest of cell cycle and suppressed cell proliferation.Additionally,changes of m6 A levels could be detected based on the CREBBP-m6 A reporter system. |
