背景:复发性自然流产(RSA),是一种危险的妊娠相关疾病,是妇科和产科社区争论的话题。这项研究的目的是确定RSA中DNA拓扑异构酶IIα(TOP2A)的功能,并阐明其潜在的分子机制。
方法:通过转染特异性sh-RNA慢病毒和过表达质粒,产生TOP2A敲低和过表达的体外模型,分别。通过在含有TOP2A抑制剂PluriSIn2的培养基中培养双细胞阶段的小鼠胚胎来建立体外TOP2A抑制模型。采用免疫组织化学染色法分析TOP2A在RSA患者绒毛组织中的表达。蛋白质印迹和qRT-PCR用于分析TOP2A的表达和参与滋养层功能的蛋白。FOXO信号通路,和植入前胚胎的发育。5-乙炔基-2'-脱氧尿苷染色,用TUNEL法和流式细胞术进一步评价TOP2A对细胞增殖和凋亡的影响。使用Transwell和伤口愈合测定来评估迁移和侵袭。此外,通过免疫荧光和线粒体相关染料测定TOP2A抑制剂对胚胎的影响。
结果:对临床样本的评估显示,与正常妊娠的妇女相比,发生RSA的患者的绒毛组织中TOP2A的表达较低(P<0.01)。体外,TOP2A敲低降低了增殖,迁移,和滋养层细胞系的入侵,细胞凋亡和FOXO信号通路激活增加(P<0.05)。相反,TOP2A过表达逆转了这些作用。此外,体内实验证实,抑制TOP2A损害滋养外胚层分化,胚胎线粒体功能以及发育速度;然而,合子基因组激活相关基因的表达没有差异。
结论:总的来说,我们的数据表明,较低的TOP2A表达与RSA有关,因为它抑制滋养层细胞增殖,迁移,和通过激活FOXO信号通路进行入侵。此外,TOP2A抑制导致小鼠植入前胚胎发育受损,这可能归因于过度的氧化应激。
Recurrent spontaneous abortion (RSA), is a dangerous pregnancy-related condition and is a subject of debate in the gynaecology and obstetrics communities. The objective of this study was to determine the function of DNA Topoisomerase II Alpha (TOP2A) in RSA and elucidate the underlying molecular mechanisms.
In vitro models of TOP2A-knockdown and -overexpression were generated by transfecting specific sh-RNA lentivirus and overexpression plasmid, respectively. An in vitro TOP2A inhibition model was established by culturing mouse embryos at the two-cell stage in a medium containing PluriSIn2, a TOP2A inhibitor. Immunohistochemical staining was used to analyse expression of TOP2A in villi tissues of patients with RSA. Western blotting and qRT-PCR were used to analyse the expression of TOP2A and proteins involved in trophoblast functions, the FOXO signalling pathway, and the development of pre-implantation embryos. 5-Ethynyl-2\'-deoxyuridine staining, TUNEL assay and flow cytometry were used to further evaluate the effect of TOP2A on cell proliferation and apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. Moreover, the effect of TOP2A inhibitor on embryos was determined by immunofluorescence and mitochondrial-related dyes.
Evaluation of clinical samples revealed that the villi tissues of patients that have experienced RSA had lower TOP2A expression compared with that from women who have experienced normal pregnancy (P < 0.01). In vitro, TOP2A knockdown decreased the proliferation, migration, and invasion of trophoblast cell lines, and increased apoptosis and activation of the FOXO signalling pathway (P < 0.05). Conversely, TOP2A overexpression reversed these effects. Moreover, in vivo experiments confirmed that inhibition of TOP2A impairs trophectoderm differentiation, embryonic mitochondrial function as well as the developmental rate; however, no differences were noted in the expression of zygotic genome activation-related genes.
Collectively, our data suggest that lower TOP2A expression is related to RSA as it inhibits trophoblast cell proliferation, migration, and invasion by activation of the FOXO signalling pathway. Additionally, TOP2A inhibition resulted in impaired development of pre-implantation embryos in mice, which could be attributed to excessive oxidative stress.