Fig 2: MCM7 mediates the effects of circKIF18A on NPC senescence under oxidative stress.a The CCK-8 assay showed the effects of circKIF18A and sh-MCM7 on NPC viability (n = 3, three different donors; *P < 0.05; **P < 0.01). b The EdU assay revealed that MCM7 mediated the effects of circKIF18A on proliferation in H2O2-treated NPCs (n = 5, five different donors; scale bar: 50 µm). c and d SA-ß-gal staining assay of NPCs transfected with or without circKIF18A and sh-MCM7 lentiviruses under oxidative stress (n = 3, three different donors for three individual experiments; *P < 0.05, **P < 0.01; scale bar: 50 µm). e and f MCM7 knockdown promoted G0/G1 arrest, which was rescued by circKIF18A overexpression, in NPCs under oxidative stress (n = 3, three different donors; *P < 0.05; **P < 0.01). g The protein levels of aggrecan, collagen II, MMP-13, and MMP-3 in NPCs transfected with circKIF18A, sh-MCM7, and empty lentiviruses were determined using western blotting (n = 3, three different donors). h The qRT-PCR assay showed the effects of MCM7 on the mRNA expression of ACAN, COL2A1, MMP13 and MMP3 in circKIF18A-overexpressing NPCs (n = 3, three different donors for three individual experiments; NS, no significance; *P < 0.05; **P < 0.01). i The protein levels of p53, p21, and p16 were measured using western blotting. NPCs were stably transfected with circKIF18A, sh-MCM7, and empty lentiviruses (n = 3, three different donors). j Western blot assays showed the effects of MCM7 on the protein levels of cyclin D1, CDK4, p-RB and RB in NPCs treated as described above (n = 3, three different donors). k The effect of p53 knockdown on the p21 level in sh-circKIF18A NPCs (n = 3, three different donors). l and m Representative images of the DCFH-DA staining assay in NPCs with circKIF18A overexpression or MCM7 knockdown under oxidative stress (n = 5, five different donors; NS no significance. Scale bar: 20 µm). All data are presented as the mean ± SD values.

Fig 3: The MCM7 protein level is increased by circKIF18A in NPCs.a Schematic illustration of the process for identifying RBPs binding to circKIF18A. b The ranking list of the top ten proteins captured by biotinylated antisense oligonucleotides (ASOs) complementary to circKIF18A. c The interaction of circKIF18A with MCM7 was verified using RIP with an anti-MCM7 antibody and IgG in NPCs (n = 3, three different donors for three individual experiments; NS, no significance; *P < 0.05; **P < 0.01). d CircKIF18A regulated the protein level of MCM7 in NPCs (n = 3, three different donors). e The mRNA expression of MCM7 in NPCs treated as described above was detected by qRT–PCR (n = 3, three different donors for three individual experiments; NS, no significance; *P < 0.05; **P < 0.01). f and g Western blot analysis of MCM7 in circKIF18A-silenced NPCs at 2 h intervals under CHX (20 µM)-mediated protein synthesis inhibition (n = 3, three different donors; *P < 0.05; **P < 0.01). h and i The effect of circKIF18A overexpression on the MCM7 level in NPCs treated with CHX at 2 h intervals (n = 3, three different donors; *P < 0.05; **P < 0.01). j Representative immunofluorescence images of NPCs transfected with sh-circKIF18A #1, sh-circKIF18A #2, circKIF18A, and empty vector lentiviruses (n = 5, five different donors; scale bar: 10 µm). k The proteasome inhibitor MG132 abolished the effect of sh-circKIF18A on reducing the MCM7 protein level in NPCs (n = 3, three different donors). l and m The ubiquitination level of MCM7 in circKIF18A-silenced or circKIF18A-overexpressing NPCs treated with MG132 (n = 3, three different donors). All data are presented as the mean ± SD values.