Abnormal Neddylation-Mediated P21 Accumulation Participates In The Pathogenesis Of Recurrent Spontaneous Abortion

Background:Recurrent spontaneous abortion(RSA)in defined as two or more consecutive pregnancy loss of a clinically established intrauterine pregnancy.It is one of the most complicated pregnancy-related diseases affecting 2%–5% of couples.However,almost half of cases are diagnosed without an exact etiology and most cases occur during the first trimester of pregnancy.Accumulating evidence has demonstrated that abnormal trophoblast development is involved in the pathogenesis of RSA.However,the underlying mechanism has not been elucidated yet.NEDD8(neural precursor cellexpressed developmentally downregulated 8)is a ubiquitin like protein,it can bind to Cullin RING ligase(CRL)complex,the largest E3 ubiquitin ligases family,via a covalent attachment,which can activate the CRL complex and further trigger the ubiquitinmediated degradation of CRL substrate.This process is called neddylation.Recently,several members cullin family,the scaffold proteins of CRL complex,have been reported to be essential for proper trophoblast development.However,the role of cullin neddylation in trophoblast development and its alteration during the pathogenesis of recurrent spontaneous abortion have not been studied yet.Method:First trimester human placental tissues of RSA patients and healthy controls(HCs)were collected from women undergoing dilatation and curettage.Western blot(WB)and immunofluorescence(IF)were utilized to analyze the expression of NEDD8 and its downstream proteins in human first trimester placenta.Immunohistochemistry(IHC)of Ki67 and in situ β-galactosidase activity assay was performed for the analysis of cell proliferation and senescence status of villus tissues.For further mechanism study,MLN4924 was used to inhibit neddylation in cells and primary cultured tissues.Cell counting kit 8(CCK8),and IF of Ki67 and p-H3 were used to evaluate Jeg-3 cell proliferation activity.Trophoblast stem(TS)cells derived from first trimester placenta were selected and cultured for the investigation of trophoblast differentiation.IF and flow cytometry were used to confirmed the purity of selected and cultured cells.Directed differentiation assays were performed,which proved the TS cells have the capacity to give rise to both extravillous trophoblast(EVT)and syncytiotrophoblast(ST)trophoblast lineages.Results:The expression pattern of NEDD8 protein was examined in HC first trimester villus.In the floating villus,NEDD8 was prominently expressed in the single layer of CTB cells;In the anchoring villus,the most intensive expression of NEDD8 was observed in the proximal cell column trophoblasts(p CCTs).Because CTBs and p CCTs highly proliferating cells,this result demonstrated that NEDD8 expression was more intensive in cells with a higher proliferation potential.Subsequently,we found neddylation inhibition related NEDD8 cytoplasm accumulation in the EVT progenitors,as well as the downregulated expression of neddylated cullins(NEDD8-cullin)in the RSA villus.Among different cullins,inhibition of CUL1 neddylation was the most significant.Moreover,a CUL1 substrate p21 was accumulated in the RSA trophoblast cells.Consistently,RSA trophoblast cells exert attenuated proliferation and early onset of senescence.A NAE inhibitor MLN4924 and si RNA were used for further mechanism study in trophoblast cells.MLN4924 treatment suppressed proliferation,caused G2 arrest and apoptosis in the trophoblast cell line Jeg-3 cells,and p21 silencing can largely alleviate the above impairments.These results indicated neddylation inhibition induced by MLN4924 attenuated proliferation,caused G2/M arrest in Jeg-3 cells via p21 accumulation.Moreover,we derived trophoblast stem(TS)cells form HC villus for trophoblast differentiation study: Upon MLN4924 treatment,the EVT differentiation was impaired as the expression of EVT specific genes such as HLA-G were downregulated and the CTB specific gene CDH1 was upregulated.In the meantime,a critical transcription factor that regulate the plasticity of trophoblast cells,GATA3,was also downregulated by neddylation inhibition.Silencing of p21 partially restored HLA-G and GATA3 levels upon MLN4925 treatment.Together with the result that cell senescence can be alleviated by p21 si-RNA,the result Indicated that,p21 accumulation caused trophoblast senescence which could affect the plasticity of trophoblast cells.ConclusionCullin1 neddylation mediated p21 degradation is required for trophoblast proliferation and can regulated trophoblast plasticity through regulating HLA-G and GATA3 expression.The results of the present study give insight to the pathological mechanism of RSA as well as the biological regulation of trophoblast development.