Research Paper Volume 13, Issue 3 pp 3726—3741

Figure 1. miR-6784 binds to and silences SphK1 in skin SCC cells. microRNA-6784 (miR-6784) putatively targets SphK1 3’-UTR at position 113-120 (A). The subcellular localization of miR-6784 was tested by qPCR, with results normalized to total miR-6784 level (B). Association between the biotinylated-miR-6784 or the biotinylated-miR-155 with SphK1 mRNA was tested by RNA-Pull Down assays in A431 cells (C). Stable A431 cells expressing the lentiviral construct encoding pre-miR-6784 (“OE-miR-6784-sLine1/2”, two lines) or the nonsense control microRNA sequence (“miRC”) were established. Expression of miR-6784, SphK1 and SphK2 was examined (D, F, G, H), with the relative SphK1 3’-UTR luciferase activity tested as well (E). Cellular ceramide contents were shown (I). A431 cells were transfected with 500 nM of wild-type (WT-) or mutant miR-6784 mimics (“Mut1-/-2”, sequences listed in J), and the control cells were transfected with nonsense control miRNA mimic (“C”); After 48h, SphK1 mRNA (K) and protein (L) expression was tested. 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. “miRC”/“C” cells.