The DNMT1/miR-34a/FOXM1 Axis Contributes to Stemness of Liver Cancer Cells

Figure 2

Effects of DNMT1 shRNA on stem-like features of MHCC97H-derived LCSCs. (a) DNMT1 protein amounts in LCSCs transfected with DNMT1 shRNA, with β-actin as a loading control. (b) miR-34a-5p levels in LCSCs transfected with DNMT1 shRNA. (c) miR-34a promoter methylation in LCSCs transfected with DNMT1 shRNA. ((d) and (e)) Representative images of spheres and colonies in LCSCs transfected with DNMT1 shRNA (left) (Scale bar, 200 μm); sphere formation efficiencies and colony formation rates were determined (right). (f) CD133 expression in LCSCs transfected with DNMT1 shRNA. (g) CD44 protein amounts in LCSCs transfected with DNMT1 shRNA. (h) Bmi1, Sox2, and Oct4 mRNA amounts in LCSCs transfected with DNMT1 shRNA. vs. LCSC (n = 3); vs. LCSCs transfected with NC shRNA (n = 3). (i) Images of subcutaneous xenografts of LCSCs (1 × 10⁵) expressing red fluorescent protein (RFP) transfected with sh NC or DNMT1 shRNA; (ii) and (iii) comparison of tumor growth curves and weights of tumors from LCSCs expressing RFP transfected with sh NC and DNMT1 shRNA. vs. sh NC group (n = 6). (iv) Micrographs of H&E staining (×200) and immunohistochemistry (×400) obtained under an optical microscope.