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 5

The functional difference between circ_0040039 and circ_0004354 in NPCs in response to TNF-α treatments. (A) The schematic illustration displays the basic characterization of circ_0040039 and circ_0004354. The red arrow represents the back-splicing site of circ_0040039. (B) Relative expression levels of circ_0040039, circ_0004354, and host gene SNTB2 in NPCs were measured using qRT-PCR after treatment in the presence or absence of RNase R for 2 h. P <0.001. (C) qRT-PCR was employed to measure the circ_0040039, circ_0004354, and SNTB2 expression levels after treatment with ACT-D at different time points in NPCs. P <0.01, P <0.001. (D, F-I) NPCs were treated with TNF-α after transfected with circ_0040039/circ_0004354 or empty vector or co-transfected with circ_0040039/circ_0004354 and miR-345-3p mimic. (D)The NPCs growth rate was detected at different time points using the CCK-8 assay. P <0.05, P <0.01, P <0.001. (E) NPCs apoptosis was measured with Annexin V APC/7-AAD double staining by using a flow cytometry detection assay in response to different concentrations of TNF-α treatments. Western blot analysis of FAF1, TP73, COL2, and IL-1β (F) as well as P21, BAX, c-GSDME, and c-CASP3 (G) proteins levels. (H) qRT-PCR measured the mRNA expression levels of ACAN, COL2, and IL-1β in NPCs. P <0.05, P <0.01, p <0.001. (I) The concentration of IL-1β was detected using ELISA in NPCs. P <0.05, P <0.01. (J) TP73 and FAF1 mRNA expression levels were measured using qRT-PCR in NPCs. P <0.01, P <0.001.

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