ABSTRACT
Objective To investigate the inhibitory effect of a disintegrin and metalloprotease 12 silenced by shR?NA on self-renewal capacity of CD133 positive giloma cells. Methods The shRNA recombinant lentivirus aimed at si?lencing ADAM12 was prepared. Human glioma cells U87 were employed in this study and assigned into three groups:shRNA-ADAM12, shRNA-NCandshRNA-C. ADAM12 expression was detected at mRNA and protein level using Re?al-time quantitative-PCR and western bloting, respectively. U87 cells were cultured with stem cell culture medium, to obtain cell sphere formation in which CD133 positive glioma cells were enriched. Immunofluorescence was employed to detect the expression of ADAM12 and CD133 in cell spheres and U87 cells; Self-renewal was tested by using tumor sphere formation assay. Molecular markers for differentiated or undifferentiated cells (CD133,GFAP and Tuj1) were de?tected at protein using western blotting. Western blotting was employed to test protein expression of HES1. Results AD?AM12 shRNA significantly down-regulated the mRNA and protein expression levels of ADAM12. Compared with shRNA–C group, the relative expression levels of mRNA in shRNA-ADAM12 group and shRNA-NC group were 0.22 ± 0.03 and 0.98 ± 0.06 (F=425.37,P<0.01). The relative expression levels of protein in shRNA-ADAM12 group, shRNA-NC group and shRNA-C group were 28.72%±2.36%, 69.21%±3.92%and 69.04%±3.57%, respectively (F=145.42,P<0.01). Immunofluorescence staining showed that expression levels of ADAM12 and CD133 in cell spheres were significantly higher than those in normal cells. The number of spheres in three groups were 45.5±2.3、104.2±5.8 and 109.6±6.2, tumor sphere formation ability of shRNA-ADAM12 group was lower than that of shRNA-NC group and shRNA-C group (F=147.03,P<0.01). Compared with the shRNA-NC group and shRNA-C group, the protain expression of GFAP and Tuj1 were increased up to 166% and 146% (P<0.01) whereas the protein expression levels of CD133 and HES1 were down-regulated by 54% and 50% (P<0.01). Conclusion Knockdown of ADAM12 may suppress self-renewal ability of CD133 positive glioma cells by inhibiting the Notch pathway activity.