Fig. 3

Validation of E2F7 as a direct target of miRNA-302a/d. a, miRNA-302a and miRNA-302d target prediction using four target genes prediction programs. b, KEGG analysis of 208 common predicted target genes. c, qRT-PCR to quantify KPNA2 level in adherent and tumor spheres of HepG2 and Huh7 cells after 3 days cultured in stem cell medium containing EGF and bFGF. qRT-PCR was used to measure the mRNA level of E2F7 after treatment of miRNA-302a/d knockdown (d) or overexpression (e). f, Western blotting was used to measure the protein levels of potential target genes after treatment of miRNA-302a/d knockdown or overexpression. AKT1 was used as a positive control for miRNA-302a/d target. g, A schematic diagram of the miRNA-302 family of 3 ‘UTR in E2F7 binding site and mutation site. Luciferase activity assay of pGL3-E2F7–3’UTR reporter co-transfected with miRNA-302a (h) and -302d (i) mimic or mutational oligonucleotides in HepG2 and Huh7 cells. j, RIP-IP assays were performed to co-IP the Ago2 complexes from HepG2 and Huh7 cells transfected with either hsa-miR-302a/d mimic or negative control mimic. Real-time PCR assays were performed on RNA samples isolated from the Ago2 co-IP fractions to measure the relative enrichment of the E2F7 mRNA. Data shown are the means ± SD of three independent experiments. Statistical analyses were performed with one-way ANOVA (** = P < 0.01, *** = P < 0.001)