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Objective To observe the changes of invasion and migration patterns and integrin expression of malignant glioma cells when Rac and Rho pathways are suppressed,respectively,in 3D hydrogel.Methods Liposome mediated pCMVLifeAct-TagGFP2 plasmids were transfected into glioma U87 cells,and then,these cells were divided into NSC23766 treatment group,Y-27632 treatment group,NSC23766 and Y-27632 combined treatment group,and control group.The three treatment groups were added NSC23766 (100 nmol/mL),Y-27632 (10 nmol/mL),100 nmol/mL NSC23766 and 10 nmol/mL Y-27632,respectively;the cells in the control group were added the same amount of medium.Cells were cultured in hydrogel;the composition of round cells,spindle cells and the mesenchymal and amoeboid cell movement transition were observed under confocal microscopy.The hydrogels of each group were infused into the microslide,and the cell chemotaxis effect were recorded and the cell movement velocities and distances were calculated in the living cell workstation.Immunofluorescence was used to observe the alternation ofintegrin expression.Results U87 cells cultured in 3D hydrogel exhibited spindle-like and round-like shapes,corresponding to mesenchymal and amoeboid cell movement.As compared with that in the control group,the proportion of round cells in the NSC23766 treatment group was significantly higher,and that of spindle cells in the Y-27632 treatment group was significantly higher (P＜0.05).The conversion rate ofmesenchymal-amoeboid transition was 50.0％ in NSC23766 treatment group and amoeboid mesenchymal transition was 42.8％ in Y-27632 treatment group as compared with that in the control group,with significant differences (P＜0.05).The velocity and distance of cells cultured in 3D hydrogel decreased orderly in NSC23766 treatment group,control group,Y-27632 treatment group and combined treatment group in chemotaxis test.The immunofluorescence test showed that integrin expression in the Y-27632 treatment group was significantly higher than that in the other three groups,and that in the control group was statistically higher than that in the NSC23766 treatment group and combined treatment group (P＜0.05).Conclusions 3D hydrogel can be used as a favorable substrate for cell culture.The combination targeted inhabitation of Rac 1 and RohA pathways provides theoretical basis for anti-invasion treatment against glioma.
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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.