FTO-mediated M~6A Demethylation Regulates ERCC1 Promoting Ovarian Aging

Background:Ovarian aging can cause decreased pregnancy rate,and increased abortion rate and birth defects rate.The end of ovarian aging is the menopause,which causes perimenopausal and menopausal syndrome,accompanied by a series of serious impact on women's physical and mental health.The assisted pregnancy for women in advanced age has become a challenge in ART.The etiological studies of ovarian aging is emerging in succession,but the regulatory mechanism of its occurrence and development has not been fully clucidated.In addition,RNA m⁶A methylation has become a new field of epigenetics in recent years.M⁶A participates in the post-transcriptional regulation,including transport,splicing,stability,degradation and translation of RNA,and plays an important role in embryonic development,tumor occurrence and organ function.But the effect on ovarian aging is reported less.Objective:We amied to explore the specific role of m⁶A modification and its demethylase FTO in ovarian aging,and to identify its downstream target genes by multi-omics approach and to verify its function.This research is expected to provide a new direction for the pathogenesis of ovarian aging and provide a new target and theoretical basis for the delay and treatment strategy of ovarian aging.Method:(1)The expression level of FTO and the modification abundance of m⁶A were investigated by the granulosa cells of infertile patients with controlled ovarian stimulation(young group and elderly group)and the ovarian tissues of different week-old mice(3,8 and 32-week-old);The relation between FTO levels and ovarian function related markers was explored;(2)The KGN cells of stably knockdown FTO gene was constructed using Lentiviral Vectors.The efficiency of knockdown was verified by WB,RT-q PCR and colorimetry.The celluar model of aging was constructed by H₂O₂ method,which was used to explore the changes of FTO level and m⁶A modification abundance.RT-q PCR,WB,?-galactosidase aging staining,Ed U staining and RNA-seq were used to identify the effects and difference between FTO knockdown and H₂O₂oxidative stress on the proliferation,apoptosis,aging and the expression of sex hormone enzyme in KGN;(3)The m RNA of downstream target was preliminary screened by Me RIP-seq.The target m RNA targeted binding with FTO was verified by RNA-seq,RT-q PCR,WB and RIP.Me RIP-RT-q PCR,ribosome density gradient centrifugation test,FTO inhibitor,protease inhibitor and other experiments combined with m⁶A site prediction were used to prove that FTO mediated m⁶A to inhibit the translation activity of target m RNA;(4)Further,the expression of target m RNA of m⁶A demethylase FTO in the ovaries of different week-age mice was verified.The effects of target m RNA knockdown on the proliferation,apoptosis,aging and the expression of sex hormone enzyme in KGN was tested to clarify the role of target m RNA on ovarian aging.Result:(1)The expression level of FTO is decreased and the content of m⁶A is increased in the elderly group.Poor ovarian function was related to the low expression of FTO;(2)Sh FTO-KGN cells were constructed.The defect of FTO significantly inhibited the proliferation,apoptosis,aging,and affected the endocrine function of KGN cells;(3)The results of ribosome density gradient centrifugation showed that the local translation activity of ERCC1 m RNA was inhibited after FTO knockdown.The use of protease inhibitors showed that the decrease of ERCC1 protein level was not reversed.FTO inhibitor and Me RIP-RT-q PCR confirmed ERCC1 was the downstream target m RNA of FTO.The results showed that FTO-dependent m⁶A significantly inhibited the local translation activity of ERCC1 m RNA;(4)ERCC1 protein level was decreased in the ovaries of the aged mice.ERCC1knockdown significantly inhibited the proliferation,promoted the apoptosis and aging,and affected the endocrine function of KGN cell.Conclusion:FTO level decreased and m⁶A content increased in ovarian aging.FTO mediated m⁶A targeted binding ERCC1 m RNA and inhibited its local translation activity,which affected the proliferation and endocrine function,and eventually promoted apoptosis and aging on the ovarian granulosa cells.This study may provide a new molecular marker for the ovarian aging.