中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2013年
6期
450-453
,共4页
吕凡%张弛%袁晓军%邬文杰%吴晔明
呂凡%張弛%袁曉軍%鄔文傑%吳曄明
려범%장이%원효군%오문걸%오엽명
原癌基因蛋白质c-myc%神经母细胞瘤%细胞增殖%细胞凋亡
原癌基因蛋白質c-myc%神經母細胞瘤%細胞增殖%細胞凋亡
원암기인단백질c-myc%신경모세포류%세포증식%세포조망
Proto-oncogene proteins c-myc%Neuroblastoma%Cell proliferation%Apoptosis
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-myc表达对细胞增殖、迁移的影响及可能机制.方法 设计构建MYCN siRNA;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(colorimetric bromodeoxyuridine)试剂盒检测细胞增殖能力变化,Transwell(Costar)系统检测细胞迁移能力变化；Westem-blot法检验RACK1、Src、Akt、ERK1/2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-myc mRNA表达(0.34±0.06)与对照组比较,差异有统计学意义(P＜0.05).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-myc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(0.68±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RACK1(对照组1.46±0.18 vs N-myc RNAi组1.13±0.10)和磷酸化Src(Tye416)(对照组1.34±0.11 vs N-myc RNAi组0.72±0.23)表达降低,磷酸化Akt表达增加(对照组0.55±0.12 vs N-myc RNAi组0.84±0.15),差异有统计学意义(P＜0.05).磷酸化Src(Tye527),磷酸化ERK1/2、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RACK1、Src,Akt的表达或活化有关.
目的 MYCN基因高擴增是神經母細胞瘤(neuroblastoma,NB)預後的重要指標,其錶達蛋白N-myc在NB髮生髮展中的意義尚存在爭議.我們以MYCN基因高擴增NB細胞株SK-N-BE(2)為研究對象,探討N-myc錶達對細胞增殖、遷移的影響及可能機製.方法 設計構建MYCN siRNA;轉染NB細胞株SK-N-BE(2),採用RT-PCR法和Western-blot法檢測錶達抑製情況；抑製N-myc錶達後,用BrdU(colorimetric bromodeoxyuridine)試劑盒檢測細胞增殖能力變化,Transwell(Costar)繫統檢測細胞遷移能力變化；Westem-blot法檢驗RACK1、Src、Akt、ERK1/2及P38錶達和燐痠化改變.結果 對照組mRNA錶達值(設為1),RT-PCR檢測顯示轉染siRNA的SK-N-BE(2)細胞中N-myc mRNA錶達(0.34±0.06)與對照組比較,差異有統計學意義(P＜0.05).Western-blot檢測結果顯示,轉染siRNA的SK-N-BE(2)細胞中N-myc蛋白錶達(0.61±0.15)與對照組(1.97±0.26)比較顯著降低,差異有統計學意義(P＜0.05).設定對照組遷移、增殖能力為1,N-myc錶達降低後SK-N-BE(2)細胞的遷移能力(0.85±0.06)和增殖能力(0.68±0.05)均顯著下降,差異有統計學意義(P＜0.05).伴隨細胞的增殖和遷移能力下降,齣現RACK1(對照組1.46±0.18 vs N-myc RNAi組1.13±0.10)和燐痠化Src(Tye416)(對照組1.34±0.11 vs N-myc RNAi組0.72±0.23)錶達降低,燐痠化Akt錶達增加(對照組0.55±0.12 vs N-myc RNAi組0.84±0.15),差異有統計學意義(P＜0.05).燐痠化Src(Tye527),燐痠化ERK1/2、燐痠化p38的錶達無顯著變化,差異無統計學意義(P＞0.05).結論 在MYCN高擴增、高錶達NB細胞株SK-N-BE(2)中N-myc錶達下降可抑製腫瘤細胞增殖和遷移,其機製可能同RACK1、Src,Akt的錶達或活化有關.
목적 MYCN기인고확증시신경모세포류(neuroblastoma,NB)예후적중요지표,기표체단백N-myc재NB발생발전중적의의상존재쟁의.아문이MYCN기인고확증NB세포주SK-N-BE(2)위연구대상,탐토N-myc표체대세포증식、천이적영향급가능궤제.방법 설계구건MYCN siRNA;전염NB세포주SK-N-BE(2),채용RT-PCR법화Western-blot법검측표체억제정황；억제N-myc표체후,용BrdU(colorimetric bromodeoxyuridine)시제합검측세포증식능력변화,Transwell(Costar)계통검측세포천이능력변화；Westem-blot법검험RACK1、Src、Akt、ERK1/2급P38표체화린산화개변.결과 대조조mRNA표체치(설위1),RT-PCR검측현시전염siRNA적SK-N-BE(2)세포중N-myc mRNA표체(0.34±0.06)여대조조비교,차이유통계학의의(P＜0.05).Western-blot검측결과현시,전염siRNA적SK-N-BE(2)세포중N-myc단백표체(0.61±0.15)여대조조(1.97±0.26)비교현저강저,차이유통계학의의(P＜0.05).설정대조조천이、증식능력위1,N-myc표체강저후SK-N-BE(2)세포적천이능력(0.85±0.06)화증식능력(0.68±0.05)균현저하강,차이유통계학의의(P＜0.05).반수세포적증식화천이능력하강,출현RACK1(대조조1.46±0.18 vs N-myc RNAi조1.13±0.10)화린산화Src(Tye416)(대조조1.34±0.11 vs N-myc RNAi조0.72±0.23)표체강저,린산화Akt표체증가(대조조0.55±0.12 vs N-myc RNAi조0.84±0.15),차이유통계학의의(P＜0.05).린산화Src(Tye527),린산화ERK1/2、린산화p38적표체무현저변화,차이무통계학의의(P＞0.05).결론 재MYCN고확증、고표체NB세포주SK-N-BE(2)중N-myc표체하강가억제종류세포증식화천이,기궤제가능동RACK1、Src,Akt적표체혹활화유관.
Objective To investigate the roles of N-myc in migration and proliferation of neuroblastoma (NB).Methods The N-myc siRNA was transfected to NB cell line SK-N-BE(2).The efficiency 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 colorimetric BrdU assay.The expressions of RACK 1,N-myc,phospho-Src (Tyr416),phospho-Src (Tyr527),phospho-Akt,phosphoERK1/2 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 expressions of RACK1 (Control group 1.46 ± 0.18 vs RNAi group 1.13 ± 0.10) and phospho-Src(Tye416)(Control group 1.34 ± 0.11 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 ± 0.1535)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.
