Figure 4

Downregulation of snoRA42 reduces in vitro tumorigenesis of CD133+ cells isolated from NSCLC cells. (A) snoRA42 knockdown inhibited migration rate of H-1944- and Calu-1-derived CD133+ cells. A wound scratch assay was used to compare the migration rate of cells treated with mock transfection, scrambled siRNA, or siRNA-snoRA42. The cells treated with siRNA-snoRA42 had a significantly higher level of migration rate compared with the cells treated with mock transfection or scrambled siRNA. (B) downregulation of snoRA42 moderated matrigel barrier invasion of H-1944- and Calu-1-derived CD133+ cells. Matrigel barrier invasion capability was compared among three different groups of CD133+ cells treated with mock, scrambled siRNA, or snoRA42-siRNA. (C) snoRA42 knockdown significantly inhibited colony formation capability of H-1944- and Calu-1-derived CD133+ cells. (D) snoRA42 knockdown induced apoptosis. Apoptosis of H-1944-derived CD133+ cells treated with scrambled siRNA and snoRA42-siRNA was compared by staining the cells with Annexin V/PI (propidium iodide) followed by flowcytometric analysis. The numbers indicate percentages of non-stained cells (Annexin V - PI-), early apoptotic cells (Annexin V + PI-) and late apoptotic or necrotic cells (Annexin V + PI+). (E). snoRA42A knockdown caused activation of caspase 3 in H-1944- and Calu-1-derived CD133+ cells. ELISA assay was used to determine cleaved caspase 3 protein in CD133+ cancer cells treated with mock transfection, scrambled siRNA, or siRNA-snoRA42. CD133+ cells showed a higher level of caspase 3 activation compared with CD133- cells after snoRA42 knockdown. *P values <0.05. The number of cells used in each experiment can be found in Methods.