The Role And Mechanism Of N6-methyladenosine Regulated By Demethylase FTO In Ovarian Aging

With the rapid development of economy and social progress,infertility has become an important medical as well as a social problem that plagues people.Ovarian aging mainly includes the content of diminished ovarian reserve(DOR),premature ovarian insufficiency(POI)and poor ovarian response(POR)to ovarian stimulation.It causes a poor response for follicles to endogenous hormones and exogenous drugs,a reducing the number of retrieved oocytes,a decreasing developmental potential of the oocytes(including disorders of chromosome segregation,mitochondrial dysfunction and abnormal cell polarity),which ultimately leading infertility.Ovarian granulosa cells(GCs)are one or more layers of supporting cells that surround the oocytes in follicles,playing a crucial role in every step of follicular development.There are extensive communication between GCs and oocytes in different time and space.The initiation of GCs growth indicates the initiation of follicular development,and the apoptosis of GCs partial regulates the atresia of follicles.It can be regulated by cytokines secreted by GCs for follicular development at all stages.In the process of ovarian aging,GCs are the targets of aging related factors such as reactive oxygen species(ROS)and advanced glycation end products(AGE),which play an important role in female infertility caused by ovarian aging.N6-methyladenosine(m6A)is the most abundant chemical modification in eukaryotic RNA which is widely present in m RNA and lnc RNA.This kind of modification is dynamically reversible in different stages,different tissues,and different pathophysiological states.m6 A participates in various life processes including regulating RNA expression and translation,cytoplasmic transport,RNA editing and alternative splicing,RNA stability and degradation,etc.Many studies have shown that m6 A is closely related to gametogenesis.However,things are still unclear to the function in GCs and the role in ovarian aging.In the first part of this study,firstly,we detected the m6 A abundance in GCs from ovarian aging patients and negative control samples by colorimetry,and tested the expression of enzymes of m6 A by q RT-PCR and western blotting.It was found that m6 A was abnormally increased in GCs of aged ovary and the expression of demethylase FTO was down-regulated,while the expressions of ALKBH5,METTL3,METTL4,METTL14,and KIAA1429 were not significantly changed.At the same time,we verified the specific expression of FTO in GCs by immunohistochemistry and cellular immunofluorescence.In order to investigate the function of m6 A in GCs of aged ovary,we establish cell models of GCs with FTO knockdown with COV434 and KGN cell lines.The dot-blot experiment showed that the abundance of m6 A in GCs increased after knocking down FTO.To reveal changes of the cells,?-galactosidase staining,and ?HA.X staining was used,showed FTO-knockdown GCs were easily to aging.At the same time,we confirmed that hydrogen peroxide,which can induce cell oxidative stress,an aging factor,can down-regulate the expression of FTO in GCs.In this section,we have initially confirmed that m6 A has abnormal expression in GCs of the aged ovary,which may be caused by the down-regulation of the demethylase FTO what certainly effect on the function of GCs and is related to the process of ovarian aging.In the second part of this study,we explored the mechanism of the effect of m6 A on GCs.In order to identify the targets erased by FTO in GCs,Methylated RNA immunoprecipitation sequencing(Me RIP-seq)and transcriptome sequencing(m RNA-seq)were performed of GCs with demethylase FTO knockdown and the control group.It was found that there were 11 molecules with increased m6 A modification and significant change in expression abundance.The samples were re-verified by Me RIP-q PCR and real-time quantitative PCR,and the quantitative relationship between the two was verified by FTO overexpression.The initially determined key target – FOS,may be critical of GCs caused by m6 A modification erased by FTO.In addition,we tested 15 pairs of samples from aged ovary and control groups by real-time quantitative PCR,which confirmed the correlation between FTO and FOS.FOS was significantly up-regulated in samples from aged ovary.In order to explore why FTO can increase the abundance of FOS-m RNA,we used actinomycin D to inhibited RNA transcription of GCs that knocked down FTO and control groups,then real-time quantitative PCR was performed to determine the abundance of FOS-m RNA,which is to investigate the effect of m6 A modification on the stability of FOS-m RNA.At the same time,we constructed a kind of plasmid with m6 A site mutated in FOS m RNA-3'UTR and the wild type plasmid as well.The GCs was transfected with the 2 kind of plasmid and the fluorescence changes were detected,which proved that FTO enhances its stability by reducing m6 A modification of FOS m RNA-3'UTR.Furthermore,we verified the function of FOS in GCs by addition and subtraction experiments.It was found that the aging caused by FTO-knockdown can be partially recovered by using small interfering RNA(si RNA)to interfere with FOS expression.Oxidative stress due to hydrogen peroxide also can up-regulate the expression of FOS in GCs.In summary,the process of ovarian aging is closely related to m6 A.This process may be caused by the down-regulated expression of the demethylase FTO in GCs,which can be ascribe to oxidative stress,which erase the m6 A modification less of FOS-m RNA-3'UTR,results FOS increases.Ultimately leading to the aging process of GCs accelerate,by what accelerates the process of ovarian aging.