Fig. 2
METTL3 induces GBAP1 expression in an m6A-dependent manner in HCC. A and B MeRIP-qPCR was conducted in METTL3 overexpressing subclones of Hep3B cells and METTL3 knockdown subclones of MHCC97H cells by using antibody against m6A or IgG. Primers of GBAP1 genes were used for qPCR. ***P < 0.001 (Student’s t test). ***P < 0.001 versus shNTC (two-way ANOVA). C and D RIP assay was conducted in Hep3B cells and MHCC97H cells. Primers of GBAP1 genes were used for qPCR. ***P < 0.001 (Student’s t test). E and F Hep3B or MHCC97H was treated with actinomycin D (10 μg/mL), an inhibitor of RNA polymerase elongation. RT-qPCR analysis was conducted to test GBAP1 expression level at 0, 2, 4, 6, 8 h after the treatment. **P < 0.01 (two-way ANOVA with Sidak’s t test). G The potential m6A site positions of GBAP1 were predicted by SRAMP platform (http://www.cuilab.cn/sramp). Four potential m6A sites with very high confidence (446A, 451A, 10631A and 10709A) were identified. H The plasmids with the A-to-G mutation at the 446, 451, 10,631 and 10,709 base of GBAP1-WT (wild type) were constructed. I and J MeRIP-qPCR was conducted in different GBAP1-mutated GBAP1-WT subclones of Hep3B and MHCC97H cells. Primers of GBAP1 genes were used for qPCR. ***P < 0.001 versus WT (two-way ANOVA). K and L RIP assay was conducted in Hep3B cells and MHCC97H cells. Primers of GBAP1 genes were used for qPCR. ***P < 0.001 versus IgG (two-way ANOVA). M–P IGF2BP2 knockdown subclones of Hep3B and MHCC97H cells were constructed. RT-qPCR and western blot were applied to test the efficiency. ***P < 0.001 versus shNTC (two-way ANOVA). Q and R RT-qPCR analysis was used to test the effect of IGF2BP2 knockdown on GBAP1 expression in Hep3B or MHCC97H cells. ***P < 0.001 versus shNTC (two-way ANOVA)