Kourdova Lucille T, Miranda Andrea L, Racca Ana C, Rojas Maria L, Del Puerto Mariano Cruz, Castro Claudia, Genti-Raimondi Susana, Panzetta-Dutari Graciela M
Universidad Nacional de Córdoba, Facultad de Ciencias Químicas, Departamento de Bioquímica Clínica, Ciudad Universitaria, X5000HUA, Córdoba, Argentina; Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Ciudad Universitaria, X5000HUA, Córdoba, Argentina.
Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Instituto de Medicina y Biología Experimental de Cuyo (IMBECU) and Universidad Nacional de Cuyo, Facultad de Ciencias Médicas, Centro Universitario, 5500, Mendoza, Argentina.
Placenta. 2022 Sep;127:62-72. doi: 10.1016/j.placenta.2022.08.002. Epub 2022 Aug 11.
Placental extravillous trophoblasts play a crucial role in the establishment of a healthy pregnancy. Reactive oxygen species (ROS) may contribute to their differentiation and function as mediators in signaling processes or might cause oxidative stress resulting in trophoblast dysfunction. The krüppel-like transcription factor 6 (KLF6) regulates many genes involved in essential cell processes where ROS are also involved. However, whether KLF6 regulates ROS levels has not been previously investigated.
KLF6 was silenced by siRNAs in HTR8-SV/neo cells, an extravillous trophoblast model. Total and mitochondrial ROS levels, as well as mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. The expression of genes and proteins of interest were analyzed by qRT-PCR and Western blot, respectively. Cell response to oxidative stress, proliferation, viability, morphology, and migration were evaluated.
KLF6 downregulation led to an increase in ROS and NOX4 mRNA levels, accompanied by reduced cell proliferation and increased p21 protein expression. Catalase activity, 2-Cys peroxiredoxin protein levels, Nrf2 cytoplasmic localization and hemoxygenase 1 expression, as well as mitochondrial membrane potential and cell apoptosis were not altered suggesting that ROS increase is not associated with cellular damage. Instead, KLF6 silencing induced cytoskeleton modifications and increased cell migration in a ROS-dependent manner.
Present data reveal a novel role of KLF6 on ROS balance and signaling demonstrating that KLF6 downregulation induces an increase in ROS levels that contribute to extravillous trophoblast cell migration.
胎盘绒毛外滋养层细胞在建立健康妊娠过程中发挥着关键作用。活性氧（ROS）可能在其分化过程中起作用，并作为信号传导过程中的介质，或者可能导致氧化应激，从而导致滋养层细胞功能障碍。类krüppel转录因子6（KLF6）调节许多参与基本细胞过程的基因，而这些过程中也涉及ROS。然而，此前尚未研究KLF6是否调节ROS水平。
在绒毛外滋养层细胞模型HTR8-SV/neo细胞中，通过小干扰RNA（siRNA）使KLF6沉默。通过流式细胞术分析总ROS水平、线粒体ROS水平、线粒体膜电位和细胞凋亡情况。分别通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法分析感兴趣的基因和蛋白质的表达。评估细胞对氧化应激的反应、增殖、活力、形态和迁移情况。
KLF6下调导致ROS和NOX4 mRNA水平升高，同时细胞增殖减少，p21蛋白表达增加。过氧化氢酶活性、2-半胱氨酸过氧化物酶蛋白水平、Nrf2细胞质定位和血红素加氧酶1表达，以及线粒体膜电位和细胞凋亡均未改变，这表明ROS增加与细胞损伤无关。相反，KLF6沉默以ROS依赖的方式诱导细胞骨架修饰并增加细胞迁移。
目前的数据揭示了KLF6在ROS平衡和信号传导方面的新作用，表明KLF6下调会导致ROS水平升高，进而促进绒毛外滋养层细胞迁移。
