K-Ras-ERK1/2 accelerates lung cancer cell development via mediating H3^K18ac through the MDM2-GCN5-SIRT7 axis

Figures & data

Figure 1. H3^K18ac expression was reduced by the Ras-ERK1/2 pathway. NCI-H2126 cells were transfected with pEGFP-N1, pEGFP-K-Ras^WT (Ras^WT), pEGFP-K-Ras^G12V/T35S (Ras^G12V/T35S) plasmids. (A and B) The expression levels of H3^K18ac, Ras and the factors of AKT and ERK pathways were then measured by western blots. Data presented as mean + SD, ** p < 0.01 (n = 3).

Figure 2. H3^K18ac was involved in regulating the functions of Ras-ERK1/2 signaling pathway in lung cancer cell phenotypes. NCI-H2126 cells were transfected with pEGFP-N1, pEGFP-H3, pEGFPH-Ras^G12V/T35S, or pEGFP-H3^K18Q (0.5, 1 and 2 μg) (indicated as GFP, H3, Ras and H3^K18Q, respectively) plasmids. (A) Cell viability, (B) numbers of colonies and (C) cell migration were determined by MTT assay, soft agar formation, and Transwell assays, respectively. Data presented as mean + SD, **p < 0.01 (n = 3).

Figure 3. The transcription of Ras-ERK1/2-targeted genes was mediated by H3^K18ac. (A) The mRNA levels of ERK1/2 downstream genes (CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16) were tested via RT-PCR after transfection with the corrective plasmids. (B) The transcription of these above-mentioned genes was detected through using ChIP after transfection with the related plasmids. Data presented as mean + SD, **p < 0.01 (n = 3).

Figure 4. Knockdown of SIRT7 adjusted H3^K18ac expression and mediated Ras-ERK1/2-affecting lung cell phenotypes. (A) After si-SIRT7-1 and si-SIRT7-2 plasmids transfection, the expression levels of SIRT7, Ras, AKT and ERK1/2 pathway associated factors were detected through RT-PCR and western blot. NCI-H2126 cells were co-transfected with pEGFP-K-RasG^12V/T35S or pEGFP-N1 plasmids and SIRT7-specific siRNA or control siRNA as indicated (Ras, GFP, si-SIRT7-1, si-SIRT7-2, or si-con, respectively). (B) Cell viability, (C) cell migration, (D) cell cycle progression and (E) ERK1/2 downstream genes were examined by MTT assay, Transwell, flow cytometry and RT-PCR assays. Data presented as mean + SD, *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3).

Figure 5. Ras-ERK1/2 activation degraded GCN5 to decline H3^K18ac expression. (A) NCI-H2126 cells were transfected with pEGFP-K-Ras^G12V/T35S, pEGFP-N1, and GCN5-HA or SIRT7-HA plasmids. After transfection, the mRNA levels of GCN5 and SIRT7 were detected by RT-PCR. (B-D) The protein levels of GCN5, SIRT7, H3^K18ac, Ras and AKT and ERK1/2 pathways associated factors were analyzed by western blot. (E) The recruitment of GCN5 to the ERK1/2 downstream genes was analyzed via ChIP analysis. (F) After treatment with MG132 (25 μM), the GCN5, Ras, and AKT and ERK1/2 pathways associated factors protein levels were analyzed by western blot with antibodies against HA and GFP. (G-H) After 24, 48, 51, 54, and 60 h transfection without MG132 treatment or 48 h transfection with MG132 treatment (25 μM, 0, 3, 6, and 12 h), the protein levels of H3^K18ac, Ras and AKT and ERK1/2 pathways associated factors were analyzed by western blot. Data presented as mean + SD, *p < 0.05, **p < 0.01 (n = 3).

Figure 6. Ras-ERK1/2 pathway-induced degradation of GCN5 was mediated by MDM2. (A-B) NCI-H2126 cells were co-transfected with GCN5-HA, pEGFP-K-Ras^G12V/T35S, of pEGFP-N1 plasmids and increasing amounts of MDM2-His plasmid (0.5, 1, and 2 μg), the protein levels of GCN5, MDM2, Ras, and AKT and ERK1/2 pathways associated factors were examined by western blot. (C-D) After transfection with a mutation in MDM2 (MDM2-MU), GCN5 and Ras protein levels were detected by western blot. (E and F) After pEGFP-N1, pEGFP-K-Ras^G12V/T35S or si-MDM2 transfection, MDM2, H3^K18ac and Ras protein levels were analyzed via western blot. (G) After co-transfection with pEGFP-K-Ras^G12V/T35S and MDM2-specific siRNA, H3^K18ac and Ras protein levels were determined by western blot.