Wound Healing Assay for Trophoblast Cells

The wound healing analysis was conducted according to a previous method [24 (link)]. In brief, 5 × 10⁴ HTR-8 cells were plated into individual well of a 6-well plate and cultured for 24 h. Then, the trophoblasts were treated with control siRNA, siUCA1 oligos, empty vector, or UCA1 overexpression vector. When the cells reached approximately 85% confluence, the trophoblasts were incubated with Mitomycin C (10 µg/mL, Tocris Bioscience, Bristol, UK) in DMEM/F12 medium for 2 h to eliminate cell proliferative ability. A sterile 200-μL tip was applied to make a scrape across the middle area of each well, followed by washing HTR-8 cells with iced-PBS for three times to remove floating cells or debris. Cells were then cultured in DMEM/F12 medium with 1% FBS for another 16 h. The distance between the edges of the scrape in each well were photographed with a Leica microscope and evaluated in collected photomicrographs. Cell migration was evaluated by comparison of the width of wound closure relative to the initial wound area at 0 h and 16 h.