Fig. 4

Biological role of miRNA-302a/d and E2F7 in HCC in vitro and in vivo. qRT-PCR (a) and Western blot (b) to quantify E2F7 levels in adherent and tumor spheres of HepG2 and Huh7 cells after 3 and 7 days cultured in stem cell medium containing EGF and bFGF. GAPDH was used as loading control. qRT-PCR (c) and Western blot (d) to quantify E2F7 levels in tumor spheres of HepG2 and Huh7 cells at Day 7 cultured in stem cell medium and differentiated liver cancer cells. e, Tumor sphere Assays of HepG2 and Huh7 cells before and after overexpression of E2F7. f, HepG2 and Huh7 cell counts in 96-well plate after transfection with negative control and E2F7 overexpression plasmids at the indicated day. g, HepG2 and Huh7 cell apoptosis was examined by flow cytometry analysis before and after overexpression of E2F7. Statistical analyses were performed with one-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. normal). h, Tumor sphere assays of HepG2 and Huh7 cells before and after overexpression of miRNA-302a/d and/or E2F7. i, HepG2 and Huh7 cell apoptosis was examined by flow cytometry analysis before and after overexpression of miRNA-302a/d and/or E2F7. The subcutaneous tumor capacity of HepG2 and Huh7 cells before and after overexpression of miRNA-302a (j) and miRNA-302d (k) and/or E2F7 was detected. Statistical analyses were performed with one-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. normal)