CYLD基因对神经母细胞瘤凋亡和活性氧水平及NF-κB信号的影响

Effects of CYLD gene on apoptosis,reactive oxygen species level and NF-κB signaling in neuroblastoma

目的CYLD在神经母细胞瘤高表达,但是具体研究机制尚不清楚。文章主要探讨上调CYLD基因表达对神经母细胞瘤凋亡、活性氧水平及NF-κB信号的影响。方法将SH-SY5Y细胞分为3组,即空白组、pcDNA3.1组(转染空白pcDNA3.1质粒)和pcDNA3.1-CYLD组(转染pcDNA3.1-CYLD质粒)。Western blot检测CYLD、p52、cyclinD1和Bax蛋白表达;流式细胞术分别检测细胞凋亡率及活性氧(ROS)水平;MTT法检测pcDNA3.1-CYLD转染24、48、72和96 h的细胞活力。结果Western blot结果显示,空白组、pcDNA3.1组和pcDNA3.1-CYLD组的蛋白表达分别为(0.074±0.010)、(0.089±0.012)和(0.787±0.067);pcDNA3.1-CYLD组CYLD表达明显高于pcDNA3.1组(P<0.05)。pcDNA3.1-CYLD组细胞活力(48h、72h和96h)明显低于pcDNA3.1组(P<0.05)。pcDNA3.1-CYLD质粒转染SH-SY5Y细胞,空白组、pcDNA3.1组和pcDNA3.1-CYLD组的细胞凋亡率分别为(4.41%±0.47%)、(4.23%±0.41%)和(21.36%±0.79%),pcDNA3.1-CYLD组细胞凋亡率明显高于pcDNA3.1组(P<0.05)。空白组、pcDNA3.1组和pcDNA3.1-CYLD组的ROS荧光强度分别为(64.74±1.02)、(66.51±1.26)和(94.39±1.58),pcDNA3.1-CYLD组ROS荧光强度明显高于pcDNA3.1组(P<0.05)。结论上调CYLD基因表达可能通过提高细胞ROS水平及抑制NF-κB信号通路促进神经母细胞瘤细胞凋亡。

Objective CYLD is highly expressed in neuroblastoma,but the specific research mechanism is still unclear.Here,the effect of up regulation of CYLD gene expression on apoptosis,reactive oxygen species level and NF-κB signaling was studied in neuroblastoma.Methods The expression of CYLD in human neuroblastoma cell line SH-SY5Y was up-regulated via the transfection of pcDNA3.1-CYLD plasmid.PcDNA3.1 was transfected as negative control(blank group).Cells were transfected for 48h,then subject to Western blotting assays for to the measure of the expression of CYLD,p52,cyclinD1 and Bax protein,and to flow cytometry for cell apoptosis rate and ROS level.Cell viability were detected CDNA3.1-CYLD transfected 24h,48h,72h and 96h by MTT assays.Results The expression of CYLD in SH-SY5Y cells was up-regulated after pcDNA3.1-CYLD transfection,which was significantly higher than that in the blank group(P<0.05).Compared with pcDNA3.1-CYLD group and the blank group,the cell vitality and the expression of p52 and cyclinD1 protein were significantly decreased,while the apoptosis rate,the ROS level,and the expression of Bax protein were significantly increased(P<0.05).Conclusion Upregulation of CYLD expression may promote apoptosis of neuroblastoma cells by increasing ROS level and inhibiting NF-κB signaling pathway.