Fig. 3
Knockdown of MSI2 significantly attenuated the stemness properties of CD44v6+ LCSC. a MSI2 expression levels were tested in CD44v6+ HCC cells and CD44v6- HCC cells by western blot in SNU-398 and MHCC-97 h cell lines. b Immunofluorescence images of CD44v6+ SNU-398 cells and CD44v6- SNU-398 cells for localization of MSI2 (red) and CD44v6 (green). Histogram analysis of the relative fluorescence intensity of CD44v6 and MSI2 in CD44v6+ cells and CD44v6- cells. Scale bar, 50 μm. c Representative images of spheres and histogram analysis in indicated cells. The inhibition of MSI2 decreased self-renewal property in vitro in CD44v6+ LCSCs, Scale bar, 200 μm. d and e Transwell migration and invasion assays showed that knockdown of MSI2 decreased the migration and invasion of CD44v6+ cells. Scale bar, 200 μm. f Colony formation assays showed that the ability of cell proliferation and colony formation of CD44v6+ cells was inhibited when MSI2 was down regulated. g 1 × 105 of MSI2 shRNA1 cells and the corresponding controls were injected into the left lobes of liver. Bioluminescence signals from MSI2 shRNA1 groups were weaker than that from corresponding control groups. Red arrows indicated the site of tumor formation. h Efficiency of tumor formation of MSI2 shRNA cells and corresponding control cells. Number of injected cells: 1 × 105. n = 8. Black arrow means Mock group, blue arrow means NC group, red arrow means MSI2 shRNA1 group, and orange arrow means MSI2 shRNA2 group. i The expression of cancer stemness-related genes, including Nanog, Oct4 and Sox2 in MSI2 shRNA cells compared with corresponding control. β-actin was used as a normalized control. It showed the expression of stemness-related genes were decreased when MSI2 was knockdown in CD44v6+ LCSCs. For statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, t test