Circ_0004354 might compete with circ_0040039 to induce NPCs death and inflammatory response by targeting miR-345-3p-FAF1/TP73 axis in intervertebral disc degeneration

Figure 6

Circ_0040039 might compete with circ_0004354 to adsorb miR-345-3p in NPCs. (A-C) NPCs were treated with TNF-α. (A) Colocalization of circ_0040039 and circ_0004354 in the cytoplasm of NPCs was measured by FISH assay. Circ_0040039 and circ_0004354 were labeled with Cy3 (red) and FITC (green), respectively. Nuclei were stained with DAPI (blue). Scale bar =20 μm. (B) The relationship between miR-345-3p and circ_0040039 and circ_0004354 was confirmed by the RIP experiment. Left, relative RNA levels compared with input. IgG acted as an NC. Right, the efficiency of AGO2 enrichment by anti-AGO2 was evaluated by western blot. P <0.01, P <0.001. (C) The levels of circ_0040039 and circ_0004354 in NPCs lysates captured by biotin-labeled miR-345-3p or NC probe. Relative RNA expression levels compared with NC. P <0.01, P <0.001. Schematic illustration of the putative binding sites of circ_0040039 (D) and circ_0004354 (F) on miR-345-3p and the WT and MUT vectors sequences of these circRNAs. (E, G) Luciferase reporter vectors carrying WT or MUT circ_0040039/circ_0004354 were co-transfected into HEK-293 T cells with miR-345-3p mimic or miR-345-3p inhibitor or corresponding NC. Relative luciferase activity was detected in the HEK-293 T cells. P <0.05, P <0.01, P <0.001.

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