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. 2022 Mar 17;298(4):101828. doi: 10.1016/j.jbc.2022.101828

Figure 5.

Figure 5

Effects of circAcbd6-miR-320-5p on differentiation of NSCs.A, RNAhybrid predicted miR-320-5p-binding sites in circAcbd6. B, effect of circAcbd6 on the abundance of miR-320-5p. (LV–NC) NSCs treated with NC of overexpression lentivirus; (LV-circAcbd6) NSCs treated with overexpression lentivirus of circAcbd6. C, luciferase reporter assay in HEK-293A cotransfected with Luciferase-miR-320-5p fusion and circAcbd6-Wild or circAcbd6-Mut, 72 h post-transfection. D, miR-320-5p expression in different tissues. E, miR-320-5p expression in NSCs, neurons, and astrocytes. F, expression of Map2, Neurod1, and ChAT detected by RT–qPCR. (miR-NC) NSCs treated with NC of miRNA mimic; (miR-320-5p) NSCs treated with miR-320-5p mimic; (miR-NCi) NSCs treated with NC of miRNA inhibitor; (miR-320-5pi) NSCs treated with miR-320-5p inhibitor. G, expression of Tuj1 and ChAT detected by Western blot. (miR-NC) NSCs treated with NC of miRNA mimic; (miR-320-5p) NSCs treated with miR-320-5p mimic; (LV-circ + miR-320-5p) NSCs treated with overexpression lentivirus of circAcbd6 and miR-320-5p mimic. H, the secretion level of ACh measured with ELISA. I, the percentage of Tuj1-positive cells was detected by flow cytometry assay. J, immunofluorescence analysis of Tuj1 (green) and ChAT (red) double-positive cells. Nuclei were stained with Hoechst. The bar represents 100 μm. The data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Acbd6, acyl-CoA-binding domain–containing 6; ACh, acetylcholinesterase; HEK-293A, human embryonic kidney 293A cell line; LV, lentiviral vector; NC, negative control; NSC, neural stem cell; qPCR, quantitative PCR.