Research Paper Volume 12, Issue 6 pp 5399—5410

Figure 1. MiR-3677 targets and downregulates SphK1 in human OS cells. MiR-3677 (-3p) putatively targets the 3’-UTR (untranslated region) of human SphK1 (at position 235-242) (A). RNA-Pull down assay results in primary human OS-1 cells demonstrated the direct association between biotinylated-miR-3677 and SphK1 mRNA (B). In parental control OS-1 cells (“Ctr”), stable OS-1 cells with pre-miR-3677-expressing lentivirus (“lv-pre-miR-3677”, s-L1/s-L2, two lines) or with the lentiviral non-sense control miRNA (“lvmiC”) construct, expression of mature miR-3677 (-3p, C), SphK1 mRNA (E) and listed proteins (F) were tested by qPCR and Western blotting assays, with the relative SphK1 3’-UTR activity (D) and ceramide contents (G) tested as well. OS-1 cells were transfected with 500 nM of non-sense microRNA control (“miC”), the wild-type (“WT”) or the mutant miR-3677 (-3p) mimics (sequences listed in A, “Mut1/2”), with SphK1 3’-UTR activity (H) and SphK1 mRNA (I)/protein (J) expression tested after 48h. Furthermore, SphK1 mRNA directly binds to biotinylated-WT miR-3677, but not to the mutants (“Mut1/2”, -biotinylated) in OS-1 cells (K). U2OS and MG63 cells as well as primary human OS cells (OS-2 and OS-3) were infected with lv-pre-miR-3677 or lvmiC, after 48h expression of mature miR-3677 (-3p, L) and SphK1 mRNA (M) was tested. Data were presented as mean ± SD (n=5), and results were normalized. ***P< 0.001 vs. “lvmiC”/“miC” cells. Experiments in this figure were repeated five times with similar results obtained.