GOLPH3对人脑胶质瘤细胞周期及凋亡调节作用的研究

Role and mechanism of GOLPH3 in cell cycle and apoptosis regulation in human glioma

目的探讨GOLPH3(Golgi phosphoprotein 3)对人脑胶质瘤细胞周期及凋亡的影响及调控机制。方法在U251胶质瘤细胞中利用siRNA下调GOLPH3的表达后,流式细胞术检测细胞周期和凋亡的变化,RT-PCR和Western blot技术检测细胞周期相关蛋白cyclin D1,p21^(waf1/cip)(p21)和p53在核酸和蛋白水平的变化,同时检测下调GOLPH3对Akt、p Akt蛋白水平的影响。结果 3条GOLPH3 siRNA均能下调GOLPH3的蛋白表达(均P<0.05);下调GOLPH3使细胞周期阻滞在G0/G1期(P<0.05),细胞凋亡率明显增加(P<0.05)。同时发现下调GOLPH3使cyclin D1的核酸和蛋白水平降低,p21的核酸和蛋白水平升高而p53的核酸和蛋白水平无明显变化。下调GOLPH3的表达降低p Akt的蛋白水平(P<0.05),但对Akt无影响。结论在U251细胞中下调GOLPH3使细胞阻滞在G0/G1期,促进细胞凋亡。下调GOLPH3对细胞周期的调节作用可能是通过抑制PI3K-Akt信号通路,进而下调cyclin D1和上调p21的表达来实现的。

Objective To explore the effect and mechanism of GOLPH3 on cell cycle and apoptosis regulation in human glioma cells. Methods Firstly, the down-regulation effect of GOLPH3 siRNAs was detected by Western blotting analysis. Then, flow cytometry was used to detect cell cycle and apoptosis. Thereafter, the mRNA and protein expression of cyclin D1, p21^wafl/cip （p21） and p53 were detected by RT-PCR and Western blotting respectively. Finally, the level of Akt and pAkt was examined by Western blotting after GOLPH3 knocking down. Results GOLPH3 expression was significantly down-regulated by three GOLPH3 siRNAs （P 〈 0.05 ）. Depletion of GOLPH3 induced GO/G1 cell cycle arrest and promoted cell apoptosis. Meanwhile, cyclin D1 expression decreased and p21 expression increased （P 〈 0. 05 ） after GOLPH3 down-regulation. However, GOLPH3 knocking-down did not affect the expression of p53 in U251 glioma ceils. Interestingly, the level of pAkt, but not the Akt, decreased after GOLPH3 depletion. Conclusions Down-regulation of GOLPH3 induces GO/G1 cell cycle arrest and promotes cell apoptosis. The effect of GOLPH3 down-regulation on cell cycle is achieved by inhibiting PI3K-AKT signal pathway and then by decreasing cyclin D1 and increasing p21 expression.