N—myc蛋白表达对神经母细胞瘤SK-N-BE（2）细胞增殖迁移的影响

Decreased expression of N-myc protein inhibits cell proliferation and migration of neuroblastoma cell line SK-N-BE（2）

【摘要】目的MYCN基因高扩增是神经母细胞瘤（neuroblastoma，NB）预后的重要指标，其表达蛋白N-myc在NB发生发展中的意义尚存在争议。我们以MYCN基因高扩增NB细胞株SK—N-BE（2）为研究对象，探讨N-mye表达对细胞增殖、迁移的影响及可能机制。方法设计构建MYCNsiRNA；转染NB细胞株SK—N—BE（2），采用RT-PCR法和Western-blot法检测表达抑制情况；抑制N-myc表达后，用BrdU（colorimetricbromodeoxyuridine）试剂盒检测细胞增殖能力变化，Tr—answell（Costar）系统检测细胞迁移能力变化；Western-blot法检验RACKl、Src、Akt、ERKl／2及P38表达和磷酸化改变。结果对照组mRNA表达值（设为1），RT-PCR检测显示转染siRNA的SK-N-BE（2）细胞中N—mycmRNA表达（（）．34±0．06）与对照组比较，差异有统计学意义（P〈（）．05）。Western-blot检测结果显示，转染siRNA的SK—N-BE（2）细胞中N—myc蛋白表达（0．61±0．15）与对照组（1．97±0．26）比较显著降低，差异有统计学意义（P％0．05）。设定对照组迁移、增殖能力为1，N—mye表达降低后SK—N—BE（2）细胞的迁移能力（0．85±0．06）和增殖能力（O．68±0．05）均显著下降，差异有统计学意义（P〈0．05）。伴随细胞的增殖和迁移能力下降，出现RACKl（对照组1．46±0．18VSN—mycRNAi组1．13±0．10）和磷酸化Src（Tye416）（对照组1．34±0．11VSN—mycRNAi组0．72±0．23）表达降低，磷酸化Akt表达增加（对照组0．55±0．12VSN—mycRNAi组0．84±0．15），差异有统计学意义（P％0．05）。磷酸化Src（Tye527），磷酸化ERKl／2、磷酸化p38的表达无显著变化，差异无统计学意义（P〉0．05）。结论在MYCN高扩增、高表达NB细胞株SK-N—BE（2）中N-myc表达下降可抑制肿瘤细胞增殖和迁移，其机制可能同RACKl、Src，Akt的表达或活化有关。

[Abstract] Objective To investigate the roles of Nmyc in migration and proliferation of neuro blastoma （NB）. Methods The N-myc siRNA was transfected to NB cell line SK-N-BE（2）. The effi- ciency of gene silence was confirmed by real time RT-PCR and western-blot. After N-myc silencing, cell migration and proliferation were detected by transwell system and eolorimetric BrdU assay. The expressions of RACK1, N-myc, phospho-Src （ Tyr416）, phospho-Src （ Tyr527 ）, phospho-Akt, phospho- ERK1/2 and phospho p38 were analyzed by western blotting. Results When the expression of N-Myc protein was significantly reduced （0. 61 ± 0. 15 vs 1.97-± 0. 26,_P〈0. 05）,the migration （0. 85 ± 0. 06） and proliferation （0.68 ± 0.05） of SK-N-BE（2） were significantly decreased （P〈0.05）. The expres- sions of RACK1 （Control group l. 46 ± 0. 18 vs RNAi group 1.13 ± 0. 10） and phospho-Src（Tye416） （Control group 1.34 ± 0. ll vs RNAi group 0. 72 ±0. 23） were also significantly decreased （P〈 0.05） ,while the expression of phospho-Akt （Control group 0. 551 9 ± 0. 121 9 vs RNAi group 0. 8360 ± 0. 153 5）was significantly increased （P〈0. 05）. The expressions of phospho-Src（Tye527）, phospho- ERK1/2 and phospho-p38 were not changed significantly by N-Myc gene silencing in SK-N-BE（2） （P〈0. 05）. Conclusions These results suggest that the high expression of N-myc in SK-N-BE（2） is crucial for cell migration and proliferation. The expressions of RACK1, phosphorylation of Src （Tye416） and Akt might be involved in the N-myc-induced migration and proliferation of NB.