背景:已显示去泛素化的失调会影响先兆子痫(PE)的发展。解整合素和金属蛋白酶9(ADAM9)在不同的生理环境中发挥作用,包括PE。这里,本研究旨在探讨在PE过程中ADAM9是否通过泛素特异性蛋白酶22(USP22)去泛素化酶介导的去泛素化调节滋养层细胞功能障碍.
方法:通过qRT-PCR和蛋白质印迹测定法测试基因和蛋白质的水平。细胞增殖,迁移,使用细胞计数试剂盒-8,5-乙炔基-2'-脱氧尿苷(EdU)检测侵袭,流式细胞术,transwell和伤口愈合试验,分别。使用蛋白质印迹分析上皮-间质转化相关标志物。通过共免疫沉淀测定鉴定USP22和ADAM9之间的蛋白质。
结果:ADAM9在PE患者中高表达,功能上,ADAM9过表达削弱了增殖,迁移,入侵,和滋养细胞中的EMT进展。机械上,去泛素化酶USP22去除ADAM9上的泛素化并保持其稳定性。USP22的强制表达还抑制滋养层细胞的增殖和移动性。此外,USP22对滋养细胞的调节作用可通过ADAM9沉默而逆转。此外,USP22与ADAM9相互作用调节Wnt/β-catenin通路的激活。
结论:ADAM9被USP22去泛素化并稳定,然后抑制增殖,迁移,入侵,和滋养细胞的EMT进展,说明USP10/RUNX1轴在PE过程中的一条新的途径。
The dysregulation of deubiquitination has been shown to affect the development of pre-eclampsia (PE). A disintegrin and metalloprotease 9 (ADAM9) plays roles in diverse physiological contexts, including PE. Here, this study aimed to investigate whether ADAM9 regulated trophoblast cell dysfunction through ubiquitin-specific protease 22 (USP22) deubiquitinase-mediated deubiquitination during PE.
Levels of genes and proteins were tested via qRT-PCR and western blotting assays. Cell proliferation, migration, and invasion were detected using cell counting kit-8, 5-ethynyl-2\'-deoxyuridine (EdU), flow cytometry, transwell and wound healing assays, respectively. Epithelial-mesenchymal transition related markers were assayed using western blotting. Proteins between
USP22 and ADAM9 were identified by co-immunoprecipitation assay.
ADAM9 was highly expressed in PE patients, functionally, ADAM9 overexpression weakened the proliferation, migration, invasion, and EMT progression in trophoblast cells. Mechanistically, the deubiquitinase
USP22 removed ubiquitination on ADAM9 and maintained its stability. Forced expression of
USP22 also suppressed the proliferation and mobility in trophoblast cells. Moreover, the regulatory effects of
USP22 on trophoblast cells were reversed by ADAM9 silencing. In addition,
USP22 interacted with ADAM9 to regulate the activation of Wnt/β-catenin pathway.
ADAM9 was deubiquitinated and stabilized by USP22 and then suppressed the proliferation, migration, invasion, and EMT progression in trophoblast cells, indicating a new pathway of USP10/RUNX1 axis in PE process.