The Role And Mechanism Of SIAH1 Mediating TRF2 Protein Stability In Regulating Ovarian Hypofunction

ObjectiveOur results indicate that ROS induces telomere abnormalities in GCs by promoting SIAH1-mediated TRF2 degradation,which causes cellular senescence,and finally contributes to POF.MethodsIn the previous study,we successfully established a rat model and CTX-GCs of premature ovarian failure(POF).After identifying the POF model in vivo and in vitro by the general situation,the estrous cycle and hormone levels of the rats,we used DHE to detect reactive oxygen stress(ROS),SA-?-gal was used to detect aging levels,and western blot was used to detect the level of P53 and SIAH1 proteins.Different concentrations of hydrogen peroxide(H₂O₂)were treated on the human ovarian granulosa cell line(KGN)to detect the expression of P53,SIAH1 and the level of cell senescence to analyze the effect of ROS and SIAH1 on senescence.Then we detected the expression of TRF2(a substrate of SIAH1)in POF,and further explore the mechanism by which SIAH1,an E3 ubiquitination ligase,induces TRF2 ubiquitination.(1)Immunoprecipitation and colocalization showed the interaction between SIAH1 and TRF2.(2)We analyzed the effect of SIAH1 on TRF2after overexpressing or knockdown SIAH1;(3)Under the treatment of the proteasome inhibitor(MG132),we detected SIAH1 and TRF2 expression;(4)Cycloheximide was used to detect the half-life of TRF2 protein when SIAH1 overexpressed(half-life);(5)When SIAH1 gene silencing or SIAH1 overexpression,the ubiquitination analysis was performed;The protective mechanism of TRF2 on telomeres in POF model.We detected telomere length by q RT-PCR,telomere morphology by PNA-FISH,and further revealed the final factor of SIAH1 for GCs senescence in POF.Results1.Rats in POF model appeared body weight loss,decreased activity,body hair dull and decreased in eating.Meanwhile,irregular estrus cycle,decreased E2 and AMH,increased FSH and LH in the serum,increased atresia follicles,increased GCs apoptosis were also observed in POF model.2.ROS induced the increase of SIAH1 and promotes GCs senescence.In the POF and CTX-GCs models,the levels of ROS and senescence were significantly increased.At the same time,the P53 and SIAH1 proteins in POF were higher.With the increase of H₂O₂concentration,the level of P53 and SIAH1 gradually increased,as well as the senescence of GCs.3.The mechanism by which SIAH1 induces TRF2 ubiquitination in POF was revealed.The expression of TRF2 in the POF model in vivo and in vitro was negatively correlated with the expression of SIAH1 protein.SIAH1 significantly increased the ubiquitination of endogenous and exogenous TRF2.The proteasome inhibitor MG132 reduced the ubiquitination and degradation of TRF2 protein induced by SIAH1,and SIAH1 could significantly shorten the half-life of TRF2.4.The protective mechanism of TRF2 on telomeres in the POF model.Overexpression of SIAH1 will reduce the expression of TRF2 protein.At this time,we found that the telomere length increases,and the KGN telomere morphology was obviously abnormal,and the chromosome was abnormal.At the same time,overexpression of the TRF2 gene could partly save the abnormal growth of KGN telomere length and abnormal telomere morphology,and then save the senescence of KGN.ConclusionThe ROS/P53/SIAH1/TRF2 signaling pathway may be involved in GCs senescence.The results of the present study reveales that the E3 ligase SIAH1contributes to GCs senescence by regulating telomere length and morphology via TRF2,providing a novel epigenetic mechanism for POF pathogenesis.