Fig. 4

GAGE7B activates p38δ/ pMAPKAPK2/ pHSP27 pathway. a The upregualted mRNA expressions of p38δ in AGS and BGC823 cells were validated by RT-qPCR (t-test, **P < 0.01, *P < 0.05). However, the effect of GAGE7B on the expressions of MAPKAPK2, HSP27, PI3K and AKT1 was not significant (t-test, P > 0.05). b The analysis revealed that the protein expressions of p38δ, pMAPKAPK2, and pHSP27 were significantly upregualted by GAGE7B in AGS or BGC823 cells in Western-blot assay (t-test, **P < 0.01, *P < 0.05). Though, the upregualtion of pMAPKAPK2 was not significant, its expression was upregulated by 43.3% in AGS cells. However, the total protein expressions of MAPKAPK2 and HSP27 were not influenced by GAGE7B (t-test, P > 0.05). c The expressions of the genes of p38δ/ pMAPKAPK2/ pHSP27 pathway, including p38δ and pHSP27, were significantly upregulated, with the upregulation of pMAPKAPK2 by 39.8%, by GAGE7B, indicating that GAGE7B activated p38δ/ pMAPKAPK2/ pHSP27 pathway in vivo (t-test, *P < 0.05,**P < 0.01,***P < 0.001). d-g The relative luciferase activity of GAGE7B pmirGLO-3′ UTR vector was significantly decreased by miR-30c in two gastric cancer cell lines (d) (t-test, **P < 0.01, *P < 0.05). In addition, two binding sites of miR-30c in p38δ (MAPK13)-3′ UTR were predicted, and the relative luciferase activity of p38δ-3′ UTR-1 was reduced by miR-30c-1-3p and miR-30c-2-3p by 35.3 and 30.9% in MKN45 cells, and was reduced by 27.4 and 35.6% in BGC823 cells. While, the relative luciferase activity of p38δ-3′ UTR-2 was reduced by miR-30c-1-3p and miR-30c-2-3p by 19.4 and 24.8% in MKN45 cells, and was reduced by 23.1 and 21.8% in BGC823 cells (e and f). GAGE7B and p38δ protein expressions were dramatically reduced by miR-30c in Western-blot assay (g) (t-test, **P < 0.01, *P < 0.05). h and i. The relative luciferase activity of p38δ-3ÚTR-1, but not p38δ-3ÚTR-2, was enhanced after the transfection of GAGE7B-3’UTR in gastric cancer cells, compared with that in GAGE7B-3’UTR-M group (t-test, *P < 0.05) (h). The upregulation of p38δ protein by GAGE7B-3’UTR was detected in Western-blot assay. And the upregulation of p38δ, pMAPKAPK2 and pHSP27, but not MAPKAPK2 or HSP27, by GAGE7B-3’UTR was also revealed (t-test, *P < 0.05) (i). j The result of RIP assay showed that GAGE7B-3’UTR was enriched in the AGO2 pellet relative to negative control IgG by 2.8 fold and 2.5 fold in BGC823 and AGS cells, respectively. Simultaneously, p38δ-3’UTR-1 was enriched by 1.6 fold in MKN45 cells and 1.4 fold in BGC823 cells