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 3

Identification of miR-345-3p biological functions and target genes. (A,C,F,K,L) NPCs were transfected with miR-345-3p mimic or miR-345-3p inhibitor or corresponding NC. (B,D,E) NPCs were treated with TNF-α after transfected with miR-345-3p mimic or miR-345-3p inhibitor or corresponding NC. (A) The expression levels of miR-345-3p were measured using qRT-PCR in NPCs. P <0.001. (B) The NPCs growth rate was measured at the indicated time points by CCK-8 assay. P <0.05, P <0.01, P <0.001. (C) NPCs apoptosis was measured by using a flow cytometry detection assay. Representative dot plots of apoptosis were displayed after Annexin V FITC/PI dual staining. (D) qRT-PCR assay corroborated that miR-345-3p promotes ACAN and COL2 but represses IL-1β mRNA expression levels in NPCs. P <0.05, P <0.01, P <0.001. (E) Western blot analysis of the expression of ACAN, COL2, IL-1β, c-GSDME, and c-CASP3. (F) The concentration of IL-1β was detected by ELISA in NPCs. P <0.01, P <0.001. Schematic illustration of the putative binding sites of FAF1 (I) and TP73 (J) on miR-345-3p and the WT and MUT vector sequences of these mRNAs. Relative luciferase activity of FAF1 (G) and TP73 (H) were measured in the HEK-293 T cells after co-transfected WT or MUT FAF1/TP73 vectors with miR-345-3p mimic or mimic NC. P <0.01, P <0.001. (K) The mRNA expression levels of FAF1 and TP73 were measured in NPCs using the qRT-PCR assay. P <0.01, P <0.001. (L) Western blot assay confirmed that miR-345-3p repressed FAF1 and TP73 protein levels in NPCs.

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