Fig 1: Knockdown of hsa_circ_0088732 suppressed glioma growth in vivo. (A,B) Gross glioma tumor samples were taken from nude mice at 21 days after injection of LN229 cells (*P < 0.05, **P < 0.01). (C) qRT-PCR was used to analyze hsa_circ_0088732 and miR-661 expression (**P < 0.01). (D) Representative images of H&E, Ki-67, and Tunel staining assays. (E) Western blot analysis of RAB3D expression. NC: negative control, KD: hsa_circ_0088732 siRNAs, OE: hsa_circ_0088732 overexpression plasmids.
Fig 2: Knockdown of RAB3D facilitated glioma cell apoptosis and inhibited glioma cell migration, invasion, and EMT. (A) RAB3D expression in LN229 and U87-MG cells was inhibited by transfection with RAB3D siRNA, and the efficiency of RAB3D knockdown was evaluated by qRT-PCR assays (*P < 0.05, **P < 0.01, ***P < 0.001). (B,C) Cell apoptosis in RAB3D-silenced LN229 and U87-MG cells was examined by flow cytometry and Hoechst 33258 staining. (D–G) The migration and invasion capabilities of LN229 and U87-MG cells with RAB3D knockdown were assessed by Transwell assays. (H) The levels of E-cadherin, N-cadherin, and vimentin were examined by western blotting. NC: negative control, siRNA: RAB3D siRNA.
Fig 3: RAB3D served as a target of miR-661. (A) RAB3D mRNA expression in samples of normal and tumor tissues was examined by qRT-PCR. (B) Person's correlation coefficient was used to analyze the correlation between RAB3D and miR-661. (C,D) RAB3D expression was measured by qRT-PCR and western blot assays after transfection with miR-661 mimics (**P < 0.01). (E) The binding site between RAB3D and miR-661 was identified. (F) 293T cells were co-transfected with WT-RAB3D or MUT-RAB3D and miR-661 or NC; dual luciferase reporter assays were used to analyze relative luciferase activity. (G) GEPIA showing the overall survival of glioma patients. (H) RAB3D expression in glioma and adjacent non-tumor tissues was assessed by immunochemistry (online database: The human protein atlas).
Fig 4: A schematic diagram of the hsa_circ_0088732/miR-661/RAB3D axis in glioma. hsa_circ_0088732 was formed by the Lcn2 gene by cyclization, hsa_circ_0088732 negatively regulated miR-661, and RAB3D was a target gene of miR-661.
Fig 5: hsa_circ_0088732 inhibited apoptosis and accelerated the migration, invasion, and EMT of glioma cells via miR-661 and RAB3D. LN229 and U87-MG cells were transfected with miR-661 mimics, RAB3D, and hsa_circ_0088732, respectively. The cell apoptosis rates (A,B) and numbers of migrated (C,D) and invaded (C,E) cells were assessed (**P < 0.01 vs. control group; #P < 0.05 vs. mimics + EV group; $P < 0.05 vs. mimics + RAB3D + pcDNA vector group). EV, overexpression vector; pcD, pcDNA3.1 plasmid; circ, hsa_circ_0088732. Western blot assays were performed to determine the levels of E-cadherin, N-cadherin, and vimentin expression in the transfected LN229 and U87-MG cells (F).
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