Figure 4. MiR-3677 overexpression inhibits OS cell progression by targeting SphK1. Stable OS-1 cells with pre-miR-3677-expressing lentivirus (“lv-pre-miR-3677”, s-L1) were further transfected with lentiviral SphK1-expresing construct (“+wt-SphK1”), control cells were transduced with the lentiviral construct with control miRNA (“lvmiC”), expression of listed proteins was shown (A); Cells were further cultured, cell proliferation and migration were tested by EdU incorporation (B) and “Transwell” assay (C), respectively; and results were quantified (B and C). Expression of listed proteins in stable OS-1 cells with the CRISPR-Cas9-SphK1-KO-GFP construct (“koSphK1-s-L1/koSphK1-s-L2”, two lines) or control construct (“Cas-9-C”) was shown (D). The koSphK1 cells were further infected with lv-antagomiR-3677 or lv-pre-miR-3677 for 48h, with cell proliferation (E) and migration (F) tested; and results were quantified. Expression of miR-3677 (G) and SphK1 mRNA (H) in eight (n=8) different human OS tissues (“T”) and surrounding normal bone tissues (“N”) was tested. Data were presented as mean ± SD, and results were normalized. ***P< 0.001 (A–C). ***P< 0.001 vs. “Cas-9-C” cells (D). ***P< 0.001 vs. “N” tissues (G and H). Experiments in this figure were repeated five times with similar results obtained.