Fig 2: RAP2C enhances apoptosis, inflammatory cytokine levels and extracellular matrix degradation by activating ERK signaling. (A) NP cells were infected with the lentiviral plasmids carrying RAP2C shRNA or the corresponding control shRNA, transfected with the control vector or the vector carrying the complete RAP2C coding sequence for overexpression or co-treated with the RAP2C overexpression vector and the ERK inhibitor SCH772984 (10 µmol/l). The expression levels of RAP2C, ERK and β-actin, and the phosphorylation of ERK (p-ERK) were examined via western blotting. *P<0.05, **P<0.01 vs. vector; #P<0.05 vs. NC. LPS (1 µg/ml)-treated NP cells were transfected with the control vector, or the vector carrying the complete RAP2C coding sequence for overexpression or co-treated with RAP2C overexpression vector and the ERK inhibitor SCH772984 (10 µmol/l). (B) Cell apoptosis was examined via flow cytometry analysis. Levels of (C) TNF-α, (D) IL-6 and (E) IL-1β in the culture medium of the cells were tested using ELISAs. Expression levels of (F) collagen II and (G) aggrecan were determined using reverse transcription-quantitative PCR. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. LPS. LPS, lipopolysaccharide; shRNA/sh, short hairpin RNA; NC, negative control; RAP2C, Ras-related protein 2C; NP, nucleus pulposus; OE, overexpression vector; p-, phosphorylated.

Fig 3: miR-200c-3p targets RAP2C in nucleus pulposus cells. (A) Interaction of miR-200c-3p and RAP2C 3′UTR was identified via bioinformatics analysis using TargetScan. (B) NP cells were transfected with control mimic or miR-200c-3p mimic. (C) Luciferase activities of RAP2C WT and RAP2C with the miR-200c-3p-binding site mutant (RAP2C MUT) were determined using luciferase reporter gene assays. (D) mRNA expression level of RAP2C was assessed using reverse transcription-quantitative PCR in the cells. (E) Protein expression level of RAP2C was measured via western blotting. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control. ns, no significance; miR, microRNA; WT, wild-type; MUT, mutant; RAP2C, Ras-related protein 2C; UTR, untranslated region.

Fig 4: miR-200c-3p suppresses IDD by targeting RAP2C/ERK signaling. (A) NP cells were transfected with miR-200c-3p mimic, inhibitor or corresponding control, or co-treated with miR-200c-3p inhibitor and the ERK inhibitor SCH772984 (10 µmol/l). The expression levels of RAP2C, ERK and β-actin, and the phosphorylation of ERK (p-ERK) were examined via western blotting. LPS (1 µg/ml)-treated NP cells were transfected with miRNA control inhibitor or miR-200c-3p inhibitor, or co-transfected with miR-200c-3p inhibitor and the lentiviral plasmids carrying RAP2C shRNA, or co-transfected with miR-200c-3p inhibitor and the ERK inhibitor SCH772984 (10 µmol/l). *P<0.05, **P<0.01 vs. vector; #P<0.05 vs. NC. (B) Cell apoptosis was analyzed using flow cytometry. The levels of (C) TNF-α, (D) IL-6 and (E) IL-1β in the culture medium of the cells were assessed using ELISA. The expression levels of (F) collagen II and (G) aggrecan were examined using reverse transcription-quantitative PCR. (H) The regulatory pathway of miR-200c-3p in LPS-treated NP cells. Data are presented as the mean ± SD. **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. LPS. IDD, intervertebral disc degeneration; LPS, lipopolysaccharide; shRNA/sh, short hairpin RNA; NC, negative control; RAP2C, Ras-related protein 2C; NP, nucleus pulposus; p-, phosphorylated; miRNA/miR, microRNA.

Fig 5: RAP2C is a direct target of miR-496. (A) The constructed luciferase reporter plasmids containing the predicted wt or mut miR-496 binding sites on RAP2C. (B) A luciferase reporter assay was performed to measure the luciferase activity in CRC cells after co-transfection of miRNA mimics and RAP2C wt or mut. (C) CRC cells were transfected with miR-496 mimics, si-NNT-ASI or pcDNA.NNT-ASI and the levels of the indicated proteins were measured by western blotting. (D) RAP2C was overexpressed in CRC cells by transfection with pcDNA.RAP2C. (E) CRC cells were transfected as indicated and cell viability was measured by an MTT assay. CRC cells were transfected as indicated and (F) migration and (G) invasion were measured. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. miR-NC mimics or as indicated. All experiments were conducted at least three times. RAP2C, Ras-related protein Rap-2c; miR, microRNA; wt, wild-type; mut, mutant; NC, negative control; si-, small interfering RNA; pcDNA, overexpression vector; CRC, colorectal cancer; NNT-AS1, lncRNA nicotinamide nucleotide transhydrogenase antisense RNA 1.