Research Paper Volume 13, Issue 3 pp 3726—3741

Figure 5. miR-6784-induced anti-A431 cell activity is due to SphK1 silencing. The miR-6784-overexpressed A431 cells (OE-miR-6784-sLine1) were further transduced with or without an UTR-null SphK1 construct (“+SphK1-UTR null”), and stable cells were established with puromycin selection; Control cells were the parental control cells (“Ctrl”); Expression SphK1/2 and miR-6784 was shown (A, B, D); Cellular ceramide contents were tested (C); Cells were further cultured for applied time periods; Cell viability, proliferation, and apoptosis were tested by CCK-8 (E), EdU staining (F), and TUNEL staining (G) assays, respectively. Stable A431 cells with the CRISPR-Cas9-SphK1-KO-GFP construct (“KO-SphK1”) were further infected with lv-antagomiR-6784 (“antagomiR-6784”), lv-pre-miR-6784 (“OE-miR-6784”), or nonsense control miRNA sequence (“miRC”) for 48h, and puromycin was added to select stable cells. Control cells were transduced with empty vector (“Vec”); Expressions of SphK1 and miR-6784 were shown and results were quantified (H–J); Cells were further cultured for applied time periods, cell viability (K), proliferation (L), and apoptosis (M) were tested similarly. Data were presented as mean ± standard deviation (SD, n=5). Experiments in this study were repeated three times with similar results obtained. *p< 0.05 vs.“Ctrl”/“Vec” cells. ^#p< 0.05. “n.s.” stands for non-statistical difference.