Lentivirus-mediated PHLDA2 overexpression inhibits trophoblast proliferation, migration and invasion, and induces apoptosis

Abstract

Inadequate trophoblast invasion and increased trophoblast apoptosis cause serious pregnancy complications. Pleckstrin homology-like domain, family A, member 2 (PHLDA2) has been linked to fetal size at birth and growth restriction in a number of studies. However, the impact of PHLDA2 on trophoblast function had not been studied previously, to the best of our knowledge. In the present study, immunofluorescence staining demonstrated that primary trophoblasts isolated from placental villous tissues were positive for cytokeratin 18 (CK18), vimentin and human placental lactogen (hPL). JEG-3 cells and primary trophoblasts were infected with lentivirus overexpressing PHLDA2. RT-qPCR and western blot analysis detected high levels of PHLDA2. A Cell Counting Kit-8 (CCK-8) assay showed that PHLDA2 overexpression inhibited trophoblast proliferation. In addition, PHLDA2 significantly induced apoptosis, as evidenced by Annexin V-FITC/propidium iodide (PI) and Hoechst staining, along with activation of Bax and caspase-3 and also decreased Bcl-2 expression. Further investigation showed that PHLDA2 effectively induced reactive oxygen species (ROS) generation, caused cytochrome c release from the mitochondria into the cytosol and decreased mitochondrial membrane potential. PHLDA2 likely induced apoptosis through the mitochondrial pathway. Wound healing and Transwell assays indicated that PHLDA2 overexpression efficiently suppressed cell migration and invasion. These data suggest that PHLDA2 plays an important role in the occurrence and development of pregnancy complications by promoting trophoblast apoptosis and suppressing cell invasion.

Identification of Potential Crucial Genes Associated With the Pathogenesis and Prognosis of Endometrial Cancer

“…Previous studies have suggested that is a growth suppressor gene and that overexpression of this gene in the placenta leads to growth restricted pregnancies both in humans and in animal models (Jensen et al, 2014). Furthermore, ectopic expression of PHLDA2 results in pregnancy complications possibly by promoting apoptosis and suppressing trophoblast growth (Jin et al, 2016). In cancer, the role of PHLDA2 is controversial.…”

Single-cell RNA sequencing reveals regulatory mechanism for trophoblast cell-fate divergence in human peri-implantation conceptuses

“…Lentiviruses were also generated in Shanghai Taitool Bioscience Co., Ltd. (China). For TBX3 knockdown experiment, lentivirus transduction at a multiplicity of infection (MOI) of 10 was accomplished according to previous studies [50, 51]. Transfected Cells were treated with the addition of 2 μg/mL puromycin (Gibco, Grand Island, NY, A1113803) for 2 to 3 passages, and cells stably expressing GFP were used for subsequent analyses.…”

Single-Cell RNA Sequencing Reveals Regulatory Mechanism for Trophoblast Cell-Fate Divergence in Human Peri-Implantation Embryo

“…Lentiviruses were also generated in Shanghai Taitool Bioscience Co., Ltd. (China). For TBX3 knockdown experiment, lentivirus transduction at a multiplicity of infection (MOI) of 10 was accomplished according to previous studies [36, 37]. Transfected Cells were treated with the addition of 2μg/mL puromycin (Gibco, A1113803) for 2-3 passages and cells stably expressing GFP were used for subsequent analyses.…”