The study explores the role of neddylation in early trophoblast development and its alteration during the pathogenesis of recurrent spontaneous abortion (RSA). Immunofluorescence and western blot were conducted to evaluate the expression pattern of NEDD8 protein in the first-trimester placentas of healthy control and RSA patients. Neddylated-cullins, especially neddylated-cullin1, were downregulated and their substrate, p21, was accumulated in RSA samples. NEDD8 cytoplasmic recruitment was observed in extravillous trophoblast (EVT) progenitors of RSA placentas. Consistent with the results of clinical samples, neddylation inhibition using MLN4924 in trophoblast cell lines caused obvious p21 accumulation and free NEDD8 cytoplasmic recruitment. Further in vitro study demonstrated neddylation inhibition attenuated proliferation of Jeg-3 cells via p21 accumulation. Moreover, when trophoblast stem (TS) cells derived from first-trimester placentas were cultured for differentiation analyses. MLN4924 impaired the differentiation of TS cells towards EVTs by downregulating HLA-G and GATA3. p21 knockdown could partly rescue MLN4924-suppressed HLA-G and GATA3 expression. In conclusion, cullin1 neddylation-mediated p21 degradation is required for trophoblast proliferation and can affect trophoblast plasticity by affecting HLA-G and GATA3 expression. The results provide insights into the pathological mechanism of RSA and the biological regulation of trophoblast development.

中文翻译：

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通过调节滋养层细胞的增殖和分化，异常的Cullin1受体介导的p21积累参与了反复自然流产的发病机制。

这项研究探讨了在早发性自然流产（RSA）发病机理中，滋养细胞在早期滋养细胞发育及其变化中的作用。进行了免疫荧光和蛋白质印迹实验，以评估NEDD8蛋白在健康对照和RSA患者的妊娠早期胎盘中的表达模式。Neddylated-cullins，特别是Neddylated-cullin1，被下调，其底物p21积累在RSA样品中。在RSA胎盘的绒毛外滋养层（EVT）祖细胞中观察到NEDD8细胞质募集。与临床样品的结果一致，在滋养细胞细胞系中使用MLN4924抑制神经交联作用会导致明显的p21积累和游离NEDD8细胞质募集。进一步的体外研究表明，通过p21的积累，烯丙基化抑制作用抑制了Jeg-3细胞的增殖。此外，当培养源自早孕胎盘的滋养层干细胞（TS）进行分化分析时。MLN4924通过下调HLA-G和GATA3损害TS细胞向EVT的分化。p21敲低可以部分挽救MLN4924抑制的HLA-G和GATA3表达。总之，滋养层细胞增殖需要cullin1 neddylation介导的p21降解，并且可以通过影响HLA-G和GATA3表达来影响滋养层可塑性。结果提供了对RSA的病理机制和滋养细胞发育的生物学调控的见解。当培养源自早孕胎盘的滋养层干细胞（TS）进行分化分析时。MLN4924通过下调HLA-G和GATA3损害TS细胞向EVT的分化。p21敲低可以部分挽救MLN4924抑制的HLA-G和GATA3表达。总之，滋养层细胞增殖需要cullin1 neddylation介导的p21降解，并且可以通过影响HLA-G和GATA3表达来影响滋养层可塑性。结果提供了对RSA的病理机制和滋养细胞发育的生物学调控的见解。当培养源自早孕胎盘的滋养层干细胞（TS）进行分化分析时。MLN4924通过下调HLA-G和GATA3损害TS细胞向EVT的分化。p21敲低可以部分挽救MLN4924抑制的HLA-G和GATA3表达。总之，滋养层细胞增殖需要cullin1 neddylation介导的p21降解，并且可以通过影响HLA-G和GATA3表达来影响滋养层可塑性。结果提供了对RSA的病理机制和滋养细胞发育的生物学调控的见解。cullin1 neddylation介导的p21降解是滋养层细胞增殖所必需的，并且可以通过影响HLA-G和GATA3表达来影响滋养层可塑性。结果提供了对RSA的病理机制和滋养细胞发育的生物学调控的见解。cullin1 neddylation介导的p21降解是滋养层细胞增殖所必需的，并且可以通过影响HLA-G和GATA3表达来影响滋养层可塑性。结果提供了对RSA的病理机制和滋养细胞发育的生物学调控的见解。