Ras-PI3K pathway promotes osteosarcoma progression via regulating VRK1-mediated H2A phosphorylation at threonine 120

Figures & data

Figure 1. H2A^T120ph was specifically promoted by Ras-PI3K pathway activation. (A) Empty-pEGFP-N1, pEGFP-Ras^WT, pEGFP-Ras^G12V/Y40C and/or H2A^T120D or H2A^T120A plasmids were transfected into MG63 or U20S cells, respectively. The H2A^T120ph expression was measured using western blotting followed by densitometric analysis. (B) Empty-pEGFP-N1, pEGFP-Ras^WT or pEGFP-Ras^G12V/Y40C plasmid was transfected into MG63 or U2OS cells, respectively. The phosphate-AKT^Ser473 expression was measured using western blotting. (C) Empty-pEGFP-N1, pEGFP-Ras^WT or pEGFP-Ras^G12V/Y40C plasmid was transfected into MG63 or U2OS cells, respectively. The Ras expression was assessed using western blotting. H2A^T120ph: Histone H2A phosphorylated on threonine 120; PI3K: Phosphatidylinositol 3-kinase. *p < .05; **p < .01 (n = 3).

Figure 2. H2A^T120ph joined in the oncogenic effects of Ras-PI3K on osteosarcoma. pEGFP-N1, pEGFP-H2A, pEGFP-Ras^G12V/Y40C and/or pEGFP-H2A^T120A were indicated as GFP, H2A, Ras and H2A^T120A, respectively. (A) The viabilities of MG63 and U2OS cells were detected using MTT assay after transfection with GFP, H2A, Ras and/or increasing amounts of H2A^T120A (0.5, 1 or 2 μM). (B) The numbers of MG63 and U2OS cell colonies were counted after transfection with GFP, H2A, Ras and/or 2 μM H2A^T120A. (C) The migration of MG63 and U2OS cells was assessed using two-chamber transwell assay after transfection with GFP, H2A, Ras and/or 2 μM H2A^T120A. *p < .05; **p < .01 (n = 3).

Figure 3. H2A^T120D promoted the oncogenic effects of Ras-PI3K on osteosarcoma. pEGFP-N1, pEGFP-H2A, pEGFP-Ras^G12V/Y40C and/or pEGFP-H2A^T120D were indicated as GFP, H2A, Ras and H2A^T120D, respectively. (A) The viabilities of MG63 and U2OS cells were detected using MTT assay after transfection with GFP, H2A, Ras and/or 2 μM H2A^T120D. (B) The numbers of MG63 and U2OS cell colonies were counted after transfection with GFP, H2A, Ras and/or 2 μM H2A^T120D. (C) The migration of MG63 and U2OS cells was assessed using two-chamber transwell assay after transfection with GFP, H2A, Ras and/or 2 μM H2A^T120D. *p < .05; **p < .01 (n = 3).

Figure 4. H2A^T120ph regulated the transcription of Ras-PI3K-targeted genes in osteosarcoma cells. pEGFP-N1, pEGFP-H2A, pEGFP-Ras^G12V/Y40C and pEGFP-H2A^T120A were indicated as GFP, H2A, Ras and H2A^T120A, respectively. (A) The CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16 mRNA expressions in MG63 and U2OS cells were tested using real-time PCR after transfection with GFP, H2A, Ras and/or 2 μM H2A^T120A. (B) The input levels of H2A^T120ph in promoter regions of CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16 in MG63 cells were evaluated using chromatin immunoprecipitation (ChIP) after transfection with GFP, H2A and/or Ras. *p < .05; **p < .01 (n = 3).

Figure 5. Suppression of VRK1 alleviated Ras-PI3K-induced up-regulation of H2A^T120ph and suppressed osteosarcoma progression. (A) After siNC or si-VRK1 transfection, the VRK1 protein level in MG63 cells was detected using western blotting. (B) After pEGFP-N1, pEGFP-Ras^G12V/Y40C, si-VRK1 and/or siNC transfection, the H2A^T120ph expression in MG63 cells was measured using western blotting. pEGFP-N1, and pEGFP-Ras^G12V/Y40C were indicated as GFP and Ras, respectively. After GFP, Ras, siNC and/or si-VRK1 transfection, (C) the viability of MG63 cells was detected using MTT assay; (D) the migration of MG63 cells was measured using two-chamber transwell assay; (E) the cell cycle distribution of MG63 cells was assessed using flow cytometric analysis. VRK1: Vaccinia-related kinase 1. *p < .05; **p < .01 (n = 3).

Figure 6. Ras-PI3K pathway specific activation-induced up-regulation of H2A^T120ph was achieved by up-regulation of VRK1. (A, B) After pEGFP-N1 or pEGFP-Ras^G12V/Y40C transfection, the VRK1 mRNA and protein levels were detected using real-time PCR and western blotting, respectively. (C) After pEGFP-N1 or pEGFP-Ras^G12V/Y40C transfection, the VRK1 and H2A^T120ph expression in MG63 and U2OS cells were measured using western blotting. (D) The CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16 mRNA expressions in MG63 cells were tested using real-time PCR after transfection with pEGFP-N1, pEGFP-Ras^G12V/Y40C and/or siNC or si-VRK1. (E) The input levels of VRK1 in promoter regions of CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16 in MG63 cells were evaluated using chromatin immunoprecipitation (ChIP) after transfection with pEGFP-N1 or pEGFP-Ras^G12V/Y40C. VRK1: Vaccinia-related kinase 1. **p < .01 (n = 3).