Fig 1: RAD51-AS1 regulated EIF5A2 expression by sponging miR-140-3p. A and B Dual-luciferase reporter assays detected the relative luciferase activity of EIF5A2 in RAD51-AS1-overexpressing OVCAR3 cells and RAD51-AS1-knockdown SKOV3 cells. B and C RT-qPCR detection of EIF5A2 mRNA in RAD51-AS1-overexpressing OVCAR3 cells (p = 0.0042 vs. Vector) and RAD51-AS1-knockdown SKOV3 cells (p = 0.0298 vs. sh-NC) incubated with actinomycin D. C A schematic drawing of the screening procedure for candidate miRNAs (miR-154-5p and miR-140-3p). D A schematic drawing of the binding sites with miR-182-5p in RAD51-AS1 and EIF5A2, as well as their mutation sites. E Dual-luciferase reporter assays detected the relative luciferase activity in OVCAR3 cells transfected with RAD51-AS1 WT + miR-140-3p inhibitor (p = 0.0054 vs. RAD51-AS1 WT + NC inhibitor), RAD51-AS1 MUT + miR-140-3p inhibitor (ns vs. RAD51-AS1 MUT + NC inhibitor), EIF5A2 WT + miR-140-3p inhibitor (p = 0.0009 vs. EIF5A2 WT + NC inhibitor) or EIF5A2 MUT + miR-140-3p inhibitor (ns vs. EIF5A2 MUT + NC inhibitor). G and H RT-qPCR was used to analyze miR-140-3p expression in RAD51-AS1-overexpressing OVCAR3 cells (p = 0.0149 vs. Vector) and RAD51-AS1-knockdown SKOV3 cells (p = 0.0034 vs. sh-NC). J and K Relative mRNA and protein levels of EIF5A2 in OVCAR3 cells with knockdown of miR-140-3p (p < 0.0001 vs. NC inhibitor) and SKOV3 cells with overexpression of miR-140-3p (p = 0.0087 vs. NC mimic)
Fig 2: RAD51-AS1 promoted the malignant behaviors of OvCA cells by elevating EIF5A2 expression. A Volcano plot of differentially expressed genes in RAD51-AS1-knockdown SKOV3.ip cells (GSE89374; p < 0.05, |fold change|> 2). B Detection of EIF5A2 protein levels was done in RAD51-AS1-overexpressing OVCAR3 cells (p = 0.0009 vs. Vector) and RAD51-AS1-inhibiting SKOV3 cells (p = 0.0103 vs. sh-NC). C Western blotting assessed EIF5A2 protein levels in SKOV3 cells transfected with sh-NC + EIF5A2 (p = 0.0002 vs. sh-NC + Vector) or sh2-RAD51-AS1 + EIF5A2 (p = 0.0347 vs. sh2-RAD51-AS1 + Vector). D Cell colony formation ability was determined in SKOV3 cells transfected with sh-NC + EIF5A2 or sh2-RAD51-AS1 + EIF5A2 (p = 0.0335 vs. sh-NC + Vector; p = 0.0129 vs. sh2-RAD51-AS1 + Vector). E Cell migration was assessed in the above cell lines by wound-healing assays (p = 0.0018 vs. sh-NC + Vector; p = 0.0075 vs. sh2-RAD51-AS1 + Vector). F Cell invasion was determined in the above cell lines (p = 0.0007 vs. sh-NC + Vector; p = 0.0392 vs. sh2-RAD51-AS1 + Vector). H and I Protein levels of PCNA, cyclin D1, CDK4, c-caspase 3, E-cadherin N-cadherin, and Vimentin were measured in the above cell lines
Fig 3: Inhibition of RAD51-AS1 lowered SKOV3 cell growth in vivo. A Images of the xenograft tumors formed in nude mice of the control, sh-NC and sh2-RAD51-groups. B The growth curve of the xenograft tumors in the sh2-RAD51-AS1 group (p = 0.0073 vs. sh-NC). C The weight of the xenograft tumors isolated from nude mice in the sh2-RAD51-AS1 group (p = 0.0495 vs. sh-NC). D Relative expression levels of RAD51-AS1 (p = 0.034 vs. sh-NC), miR-140-3p (p = 0.0001 vs. sh-NC) and EIF5A2 mRNA (p = 0.0071 vs. sh-NC) in the xenograft tumors of the sh2-RAD51-AS1 group were detected. E Representative images of IHC staining of EIF5A2 in the xenograft tumors derived from nude mice; Relative protein levels of EIF5A2, Ki67, Bax, Bcl-2, cyclin D1, N-cadherin and E-cadherin in the xenograft tumors isolated from nude mice were detected by western blotting
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