小RNA干扰PCBP2在神经胶质瘤细胞中对P53相关基因的调节和对细胞增殖的影响

Knockdown of PCBP2 with small interfering RNA regulates P53 related genes and cell proliferation of glioma cells

目的探索多聚胞嘧啶结合蛋白2(PCBP2)表达下调对相关基因和胶质瘤细胞增殖的影响。方法 3株神经胶质瘤细胞系(T98G、U87MG和U251)各分为2组,分别转入特异性靶向PCBP2基因的小干扰RNA(siRNA)和对照siRNA,用Western blot法检测siRNA对PCBP2蛋白的抑制效果及对P53蛋白和它的两个靶基因PUMA和BAX的表达影响,同时检测P53共调节因子FHL2和同家族成员FHL1表达变化。用生物素pull-down分析PCBP2是否和FHL家族成员mRNA结合。用5-溴脱氧尿嘧啶核苷(Brd U)掺入法检测细胞增殖能力,Hoechst染色观察细胞凋亡率。结果 PCBP2 siRNA转染这3株细胞后使PCBP2蛋白表达水平明显下调(P<0.05);增加P53及它的靶基因PUMA和BAX的蛋白表达(P<0.05);抑制FHL2蛋白(P<0.05),但并不结合其mRNA的3'非编码区(3'UTR);Brd U阳性细胞减少(P<0.05);Hoechst染色阳性细胞增多(P<0.05)。结论抑制PCBP2表达可以增强胶质瘤细胞中P53通路活性,并减弱细胞增殖能力,诱导细胞凋亡。

Objective To study the effect of poly（C） binding protein 2 （PCBP2） silencing on P53 related genes and glioma cell proliferation. Methods PCBP2 gene-specific siRNA or control siRNA was transfected into three glioma cell lines （T98G, U87MG and U251 ）. The expression levels of PCBP2 protein, P53 and its target genes PUMA and BAX, P53 co-regulator FHL2 and another member of the Four-and-a-half-LIM domain （FHL） family FHL1 were detected by Western blot. The interactions between PCBP2 and FHL family members＇ mRNA were analyzed by Biotin pull-down. Cell proliferation was determined by BrdU incorporation. The apoptosis ratio of cells was measured by Hoechst staining. Results After transfection of PCBP2 siRNA, PCBP2 protein was evidently silenced ; the protein levels of P53 and its target genes PUMA and BAX were increased （ P 〈 0. 05 ） ; the protein level of FHL2 was decreased （P 〈0. 05） but its mRNA 3＇UTR didn＇t bind to PCBP2, the BrdU positive cells decreased （ P 〈 0. 05 ） ; the number of Hoechst positive cells increased （P 〈 0. 05 ）. Conclusions Knockdown of PCBP2 can enhance the activity of P53 pathway in glioma cells with the weakened ability of cell proliferation and induction of cell apoptosis.