Research Paper Volume 15, Issue 5 pp 1564—1590

Figure 7. Knockdown of miR-424 reverses shcircRBMS3-induced attenuation of cell proliferation, migration, and invasion in OS cells. (A) The expression of EIF4B and YRDC in HOS and 143B cells was detected by western blot analysis. Cells were co-transfected with shcircRBMS3 and miR-424 sponge or control vector. Data represent the mean ± SD (n = 3). (B) The mRNA expression of EIF4B and YRDC in HOS and 143B cells was detected by RT-qPCR analysis. Cells were transfected with control vector and shcircRBMS3 with or without miR-424 sponge. Data represent the mean ± SD (n = 3). * P < 0.05 (C) The expression of EIF4B and YRDC in HOS and 143B cells was detected by immunofluorescence analysis. Cells were transfected with control vector and shcircRBMS3, with or without miR-424 sponge. Data represent the mean ± SD (n = 3). Scale bars = 50 μm. (D) Proliferation of OS cells transfected with control vector and shcircRBMS3, with or without miR-424 sponge, was evaluated by the CCK-8 assay. Data represent the mean ± SD of three independent experiments. (E) miR-424 downregulation rescued the growth inhibition of circRBMS3 knockdown in OS cells, as determined by colony formation assays (details are shown in the insets). Data represent the mean ± SD (n = 3). * P < 0.05. (F) Downregulation of both circRBMS3 and miR-424 resulted in fewer apoptotic cells in OS cells, compared with circRBMS3 inhibition alone. Apoptosis rates were determined by Annexin V-FITC/PI staining and FACS. Data represent the mean ± SD (n = 3). * P < 0.05. (G) The downregulation of circRBMS3 and miR-424 on cell migration capability was evaluated by a wound-healing assay in HOS and 143B cells. Data represent mean ± SD (n = 3). * P < 0.05. Scale bar, 200 μm. (H) Effects of circRBMS3 inhibition on cell migration and invasion were eliminated by miR-424 downregulation. Migration and invasion of OS cells transfected with control vector and shcircRBMS3, with or without miR-424 sponge, were evaluated by the Matrigel™ and transwell invasion assays. Scale bars = 50 μm. (I) OS cells transfected with control vector and shcircRBMS3 with or without miR-424 sponge were cultured in soft agar for 20 days. Colonies were photographed.