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. 2013 Oct 29;109(11):2853–2863. doi: 10.1038/bjc.2013.672

Figure 7.

Figure 7

Knockdown of miR-125b induces apoptosis in glioblastoma cells independent of p38MAPK. (A) The predicted miR-125b-binding site in the 3′-UTR of p38MAPK. The miR-125b seed sequences and their predicted binding sites in the 3′-UTR of p38MAPK 3′-UTR shown in frame. The underlined sequences represented the mutation seed sequence of miR-125b in 3′-UTR of p38MAPK. (B) miR-125b targets the 3′-UTR of p38MAPK in luciferase reporter assay. HEK-293 cells were co-transfected with 125b-DP and p38MAPK full-length 3′-UTR-luciferase reporter or p38MAPK 3′-UTR mutation luciferase reporter, respectively. After incubation for 48 h, luciferase assay was performed as described in Materials and Methods to determine the expression of p38MAPK. (C) Overexpression of miR-125b downregulates p38MAPK expression in U251, U87 and C6 cells. NC-DP and 125b-DP were transfected into U87, U251 and C6 cells and the level of p38MAPK was analysed by western blot. (D) p38MAPK is activated by knock-down of miR-125b in U87 cells. 125b-AS or 125b-DP was transfected into U87(p53 KD) cells, and treated with or without UV radiation. After being cultured for 48 h, the cells were collected and total cell proteins were isolated. The total expression of p38MAPK and phosphorylation p38MAPK (P-p38MAPK) were detected by western blot. β-Actin was used as a loading control. (E) Cell apoptotic determination with Hoechst 33342 stain in U87(p53 KD) cells transfected with 125b-AS treated with or without SB203580. U87(p53 KD) cells were transfected with 125b-AS, after incubation for 24 h, p38MAPK-specific inhibitor was added. After incubation for another 48 h, the apoptotic cells were stained by Hoechst 33342 as showed by arrows. (F) The expression of p-p38MAPK and p-p53 in U87(p53 KD) cells transfected with 125b-AS treated with or without SB203580 was determined by western blot. After incubation for 24 h, cells were treated with or without p38MAPK-specific inhibitor, SB203580. After being cultured for another 24 h, cells were collected and nuclear protein was isolated. Western blot was performed to detect the expression of p-p38MAPK and p-p53. (G) Caspases 9 and 3 expression in U87(p53 KD) cells after being transfected with 125b-AS and treated with or without SB203580. Cell treatment was described as above and the expression of caspase-9, cleaved caspase-9, caspase-3 and cleaved caspase-3 were detected by western blot. The expression of β-actin was used as a loading control.