Effects Of TGFβ1 On Proliferation And Apoptosis Of Ovarian Granulosa Cells Regulated By KMT5C-H4K20me3 Axis

ObjectiveIt is of great practical significance to clarify the molecular mechanism of ovarian aging and to establish early intervention strategies.Granulosa cells are important somatic cells in the ovary.The imbalance of their proliferation and apoptosis is a primary cause of triggering follicular atresia,which can ultimately lead to premature ovarian failure or female infertility.This study observes the relationship between the transforming growth factorβ1（TGFβ1）and the premature ovarian insufficiency（POI）disease,explores the effect of TGFβ1 pathway on the proliferation and apoptosis of granulosa cells,and investigates the role of the KMT5C-H4K20me3 axis.The research reveals the epigenetic mechanism of ovarian aging and provides potential molecular targets for remodeling and protecting ovarian functions.Methods1.Follicular fluid samples from patients with clinical premature ovarian insufficiency and normal ovarian function were collected.The expression level of TGFβ1 was detected by ELISA,and that in sediment（mainly granulosa cells）was examined by Western blot;2.The mice model of premature ovarian insufficiency was established by combined intraperitoneal injection of cyclophosphamide（120 mg/kg）and busulfan（30mg/kg）.The expression of TGFβ1 in ovary was evaluated by q-PCR and Western blot,and the expression of TGFβ1 was examine by IHC.3.Mouse ovarian granulosa cells were isolated and cultured in vitro.After 48 h of transfection with TGFβ1 small interfering RNA（si-TGFβ1）,the proliferation and apoptosis of granulosa cells were detected by EDU and FITC/PI staining,respectively.The contents of Pg,E₂ and AMH in culture supernatant were determined by ELISA.4.Si-TGFβ1 was co-incubated with human ovarian granulosa cells for 48 h,and the proliferation and apoptosis of granulosa cells were observed by EDU and FITC/PI staining,respectively.Changes in apoptosis-related genes and hormone regulatory enzymes were detected by q-PCR and Western blot.5.Western blot was used to observe the expression of H4K20me3 after si-TGFβ1interfered with mouse granulosa cells and human KGN cells,and q-PCR and Western blot were used to measure the expression of regulatory enzyme KMT5C.6.KMT5C inhibitor A-196 was co-incubated with mouse granulosa cells for 48 h.Western blot was used to detect H4K20me3 expression in granulosa cells,and EDU and FITC/PI staining were used to observe cell proliferation and apoptosis.Q-PCR and Western blot were used to detect the changes in genes and hormone regulatory enzymes related to proliferation and apoptosis of granulosa cells,and the contents of Pg,E₂ and AMH in culture supernatants were determined.Results1.ELISA results showed that compared with the normal group,the amount of TGFβ1 in follicular fluid and in granulosa cell sediment of POI patients were significantly reduced;（P<0.001）2.The results of q-PCR and WB showed that the expression of TGFβ1 protein and mRNA in ovarian tissues of POI mice were also significantly decreased（P<0.01）.IHC results showed that TGFβ1 was widely distributed in the ovarian tissues of mice,and was expressed in the cytoplasm and nucleus of follicular cells at all levels;3.EDU showed that the proliferation rate of granulosa cells decreased after silting TGFβ1（P<0.05）,FITC/PI staining showed that the apoptosis of granulosa cells increased,q-PCR and WB showed that the mRNA and protein levels of Bax/Bcl2increased significantly（P<0.05）,and the mRNA levels of CYP11a1 and CYP19a1decreased significantly（P<0.01）.ELISA results showed that Pg,E₂ and AMH secretion of granulosa cells decreased significantly（P<0.05,P<0.01）;4.After TGFβ1 expression was silenced by small interfering RNA,EDU showed decreased KGN proliferation rate（P<0.01）,FITC/PI staining showed increased KGN apoptosis,q-PCR and WB results showed significantly increased Bax/Bcl2 protein and mRNA levels（P<0.05,P<0.01）.The mRNA levels of CYP11a1 and CYP19a1decreased significantly（P<0.001）.ELISA results showed that KGN secretion of Pg,E₂ and AMH decreased significantly（P<0.05,P<0.01）;5.After TGFβ1 was down-regulated,the expression of H4K20me3 protein in mouse granulosa cells and KGN cells was decreased（P<0.05,P<0.01）,and the expression of KMT5C protein and mRNA were decreased significantly（P<0.05,P<0.01）;6.A-196 inhibited KMT5C and down-regulated H4K20me3 expression,then the EDU results showed decreased proliferation rate of mouse granulosa cells（P<0.05）,FITC/PI staining showed increased apoptosis of mouse granulosa cells,q-PCR and WB results showed significantly increased Bax/Bcl2 protein and mRNA levels（P<0.05,P<0.01）,and PCNA mRNA and protein expression（P<0.05,P<0.01）.The mRNA levels of CYP11a1 and CYP19a1 decreased significantly（P<0.01）,and ELISA results showed that the secretion of Pg,E₂ and AMH in mouse granulosa cells decreased significantly（P<0.05,P<0.01,P<0.001）.Conclusions1.Premature ovarian insufficiency is closely associated with abnormal TGFβ1pathway;2.Post-translational modification of KMT5C-H4K20me3 regulatory axis plays an important role in the proliferation and apoptosis of ovarian granulosa cells and secretion of key hormones;3.TGFβ1 may affect the function of granulosa cells and cause premature ovarian failure through KMT5C-H4K20me3 axis.