Rnd3通过对p65的泛素化调节诱导胶质瘤细胞凋亡

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

目的探讨Rnd3在胶质瘤细胞凋亡中的作用及机制。方法通过Myc-Rnd3真核表达质粒转染,以Myc真核表达质粒转染作为对照,调控胶质瘤细胞株U251中Rnd3的表达;采用流式细胞术检测转染后U251细胞凋亡率;用Western blot技术检测细胞内Rnd3、p65、B细胞淋巴瘤/白血病-2(bcl-2)以及裂解的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved-Caspase-3)的表达;以Flag真核表达质粒转染作为对照,通过Flag-p65真核表达质粒转染恢复p65表达后,用Western blot技术检测其下游bcl-2以及cleaved-Caspase-3蛋白的表达;Myc-Rnd3转染后,用MG-132抑制蛋白泛素化,检测细胞内p65蛋白表达。结果经真核表达质粒转染后,Myc-Rnd3组细胞Rnd3蛋白表达量较Myc组显著提高(P<0.05);Myc-Rnd3组细胞凋亡由U251-Myc组7.28%(5.36±0.1、1.92±0.12)增加至U251-Myc-Rnd3组的19.93%(13.51±0.32、6.42±0.21),细胞凋亡率明显高于Myc组,差异有统计学意义(P<0.05);与U251-Myc组比较,U251-Myc-Rnd3组p65蛋白表达量降低,高表达的Rnd3使p65表达下调,进而引起bcl-2下调和cleaved-Caspase-3上调,差异有统计学意义(P<0.05);逆转p65蛋白功能后(p65^+,Rnd3^-)组较(p65^-,Rnd3^-)组p65表达量升高(P<0.05),bcl-2表达量升高(P<0.05),cleaved-Caspase-3表达量降低(P<0.05);(p65^+,Rnd3^+)组较(p65^+,Rnd3^-)组p65表达量差异无统计学意义;(p65^-,Rnd3^+)组较(p65^-,Rnd3^-)Rnd3表达量升高(P<0.05),p65表达量降低(P<0.05),bcl-2表达量降低(P<0.05),cleaved-Caspase-3表达量升高(P<0.05);MG-132抑制细胞内蛋白泛素化途径结果显示,随着Rnd3蛋白表达量升高,p65蛋白表达量依次降低;而在MG-132处理组,随着Rnd3蛋白表达量升高,p65蛋白表达量差异无统计学意义。结论Rnd3可能通过促进p65泛素化诱导U251胶质瘤细胞凋亡。

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.