背景:先兆子痫(PE)的主要症状,在怀孕期间出现的特定疾病,是蛋白尿和高血压。PE发生和发展的病理根源被广泛认为是胎盘滋养细胞功能异常。本研究旨在探讨胎盘特异性8(PLAC8)在滋养细胞侵袭和迁移中的特征和机制。
方法:采用Westernblot和实时定量PCR检测PLAC8和AlkB同源物5(ALKBH5)的表达。通过甲基化RNA免疫沉淀检测PLAC8mRNA的m6A水平。利用Transwell实验,检查细胞侵袭和迁移。酶联免疫吸附试验用于分析MMP-2和MMP-9的分泌水平。进行RNA下拉和RNA免疫沉淀以检测ALKBH5与PLAC8之间的结合。
结果:在PE组织和缺氧处理的HTR-8/SVneo细胞中,ALKBH5和PLAC8水平升高,PLAC8m6A甲基化水平降低。PLAC8与ALKBH5在临床组织中的表达呈正相干。此外,过表达PLAC8促进HTR-8/SVneo细胞迁移和侵袭,MMP-2和MMP-9的水平;而PLAC8的干扰降低了缺氧处理的HTR-8/SVneo细胞的迁移和侵袭,MMP-2和MMP-9的水平也是如此。此外,PLAC8mRNA的m6A修饰位点为GAACU(位置1449,位点2)。MMP-2和MMP-9水平升高,以及暴露于缺氧的HTR-8/SVneo细胞的迁移和侵袭,均由m6ASite2突变促进。此外,ALKBH5可以与PLAC8结合,减少其m6A修饰,并促进其表达。
结论:高表达的ALKBH5抑制PLAC8mRNA的m6A水平,促进PLAC8的表达,PLAC8过表达可促进缺氧诱导的HTR-8/Svneo细胞的侵袭和迁移,表明其在PE中具有潜在的保护作用。
BACKGROUND: The main symptoms of preeclampsia (PE), a specific ailment that develops during pregnancy, are proteinuria and hypertension. The pathological root of the onset and progression of PE is widely regarded as abnormal placental trophoblast cell function. This study aimed to look into the character and mechanism of Placenta-specific 8 (PLAC8) in trophoblast cell invasion and migration.
METHODS: Expressions of PLAC8 and AlkB homologue 5 (ALKBH5) were examined by western blot and quantitative real-time PCR. The m6A level of
PLAC8 mRNA was detected by methylated RNA Immunoprecipitation. Using Transwell experiments, cell invasion and migration were examined. The enzyme-linked immunosorbent assay was utilized to analyze the MMP-2 and MMP-9 secretion levels. RNA pull-down and RNA immunoprecipitation were conducted to detect the binding between ALKBH5 and PLAC8.
RESULTS: In PE tissue and hypoxia-treated HTR-8/SVneo cells, levels of ALKBH5 and PLAC8 were increased, and
PLAC8 m6A methylation levels were decreased. There was a positive correlation between
PLAC8 and ALKBH5 expression in clinical tissues. In addition, overexpressing PLAC8 promoted HTR-8/SVneo cell migration and invasion, and so as the levels of MMP-2 and MMP-9; while interference with PLAC8 reduced the migration and invasion of hypoxia-treated HTR-8/SVneo cells, and so as the levels of MMP-2 and MMP-9. Moreover, the
PLAC8 mRNA\'s m6A modification site was GAACU (Position 1449, Site 2). Increased levels of MMP-2 and MMP-9, as well as migration and invasion of HTR-8/SVneo cells exposed to hypoxia, were all facilitated by the m6A Site2 mutation. Furthermore, ALKBH5 could bind to PLAC8, reduce its m6A modification, and promote its expression.
CONCLUSIONS: High-expressed ALKBH5 inhibits the m6A level of
PLAC8 mRNA and promotes
PLAC8 expression, while PLAC8 overexpression can promote hypoxia-induced invasion and migration of HTR-8/Svneo cells, indicating its potential protective function in PE.