Rnd3 induces apoptosis of glioma cells through promoting the ubiquitination of p65

Abstract

Objective Explore the role and mechanism of Rnd3 during the process of apoptosis in glioma cells. Methods Rnd3 plasmid was transfected to regulate the expression of Rnd3 in U251 glioma cells, Myc plasmid was transfected as control. Flow cytometry was used to detect apoptosis. We detected the expression of Rnd3, p65, B cell lymphoma/leukemia-2 (bcl-2) and cleaved-Caspase-3 in glioma cells by Western blotting. Flag-p65 plasmid was used to restore p65 expression, then detected the expression of bcl-2 and cleaved-Caspase-3 by Western blotting, Flag plasmid was transfected as control. MG-132 was applied to inhibit the ubiquitination of protein after Rnd3 plasmid transfected, then detected the expression of p65 by Western blotting. Results After transfection with eukaryotic expression plasmid, the expression of Rnd3 protein in Myc-Rnd3 group was significantly higher than that in Myc group (P<0.05); the apoptotic rate of Myc-Rnd3 group increased from 7.28% (5.36±0.10, 1.92±0.12) in U251-Myc group to 19.93% (13.51±0.32, 6.42±0.21) in U251-Myc-Rnd3 group, and the apoptotic rate was significantly higher than that in Myc group (P<0.05); compared with U251-Myc group, the expression of p65 protein in U251-Myc-Rnd3 group decreased, and the expression of Rnd3 in U251-Myc group decreased. The expression of p65 was down-regulated, which resulted in the down-regulation of bcl-2 and the up-regulation of cleaved-Caspase-3 (P<0.05). After reversing the function of p65 protein (p65+ , Rnd3-), the expression of p65 was increased (P<0.05), the expression of bcl-2 was increased (P<0.05), the expression of cleaved-Caspase-3 was decreased (P<0.05); (p65+ , Rnd3+ ) the expression of p65+ , Rnd3-) was higher than that of (p65+ , Rnd3-). The expression of p65-, Rnd3-) was higher than that of (p65-, Rnd3-) Rnd3 (P<0.05), p65 expression was lower (P<0.05), bcl-2 expression was lower (P<0.05), cleaved-Caspase-3 expression was higher (P<0.05), and MG-132 inhibited ubiquitination pathway of intracellular protein, which showed that the expression of p65 decreased with the increase of Rnd3 protein expression. In MG-132 treatment group, with the increase of Rnd3 protein expression, there was no significant difference in p65 protein expression. Conclusion Rnd3 induces apoptosis of U251 glioma cells may through promoting the ubiquitination of p65. Key words: Rnd3; p65; Glioma; Ubiquitination