分化抑制因子1对鼻咽癌细胞增殖的影响及其途径

Effect and pathway of Id1 on the cell growth of nasopharyngeal carcinoma

目的探讨分化抑制因子1(Id1)对鼻咽癌细胞增殖和凋亡的影响及其机制。方法体外培养人鼻咽癌细胞NP69、CNE1。合成Id1小分子干扰RNA(siRNA),构建Id1表达载体并分别转染鼻咽癌细胞NP69、CNE1。收集Id1siRNA及对照质粒Scramble转染72h的NP69细胞(分别命名为NP69si-Id1、NP69-NC),pcDNA3.1-Id1及对照pcDNA3.1转染48h的CNE1细胞(分别命名为CNE1-Id1、CNE1-Vector),采用RT-PCR及Western blotting检测Id1的表达情况。采用MTT法比较顺铂(DDP)对NP69si-Id1、NP69-NC和CNE1-Id1、CNE1-Vector增殖的影响。采用CalcuSyn软件计算各组细胞对DDP的IC50值。将1μg/ml DDP与鼻咽癌细胞共同孵育24h后,采用Western blo ing检测凋亡蛋白Caspase-3、Bax、Bad的表达及蛋白激酶B(Akt)Thr308和Ser473的磷酸化情况。结果与NP69-NC比较,NP69si-Id1的Id1 mRNA和蛋白水平明显降低;与CNE1-Vector比较,CNE1-Id1的Id1 mRNA及蛋白水平明显升高。NP69si-Id1细胞对DDP的IC50值(0.207±0.008μg/ml)明显低于NP69-NC(0.405±0.009μg/ml,P<0.05),CNE1-Id1细胞对DDP的IC50值(0.671±0.012μg/ml)明显高于CNE1-Vector(0.445±0.008/ml,P<0.05)。Western blotting检测结果显示,与NP69-NC比较,NP69si-Id1的Caspase-3断裂带增多,Akt Thr308和Ser473磷酸化减少;与CNE1-Vector比较,CNE1-Id1的Caspase-3断裂带减少,Akt Thr308和Ser473磷酸化增加。结论 Id1可促进鼻咽癌细胞的生长,抵抗DDP诱导的细胞凋亡,其机制可能与增加Akt磷酸化有关。

Objective To investigate the effects of Id1 on proliferation and apoptosis of nasopharyngeal carcinoma（NPC） cells in vitro and its mechanism.Methods Human nasopharyngeal carcinoma cell NP69 and CNE1 were cultured in vitro.Small interfering RNA and expression vector of Id1 were transfected into NP69 and CNE1 cells respectively.NP69 cells transfected with Id1 siRNA or control plasmid Scramble for 72h（named NP69si-Id1 and NP69-NC respectively）,and CNE1 cells transfected with pcDNA3.1-Id1 and control pcDNA3.1 for 48h（named CNE1-Id1 and CNE1-Vector respectively） were collected.The effect of siRNA or pcDNA3.1-Id1 on the expression level of Id1 was evaluated by reverse transcriptase-PCR and Western blotting.MTT assay was performed to compare the effects of DDP on proliferation of both CNE1-Id1 and CNE1-Vector and both NP69si-Id1 and NP69NC.CalcuSyn software was used to calculate the IC50values of cells to cisplatin（DDP）.After the 1μg/ml DDP and nasopharyngeal carcinoma cells were coincubated for 24 hours,the expressions of cell apoptotic protein caspase-3,Bax,Bad,and the Thr308 and Ser473 phosphorylation of protein kinase B（Akt） were analyzed by Western blotting.Results Obviously lower Id1 mRNA and protein levels were detected in NP69si-Id1 cells as compared with NP69-NC cells and obviously higher Id1 mRNA and protein levels in CNE1-Id1 cells than in CNE1-Vector were found.The IC50 value of the NP69si-Id1 cells for DDP（0.207±0.008μg/ ml） was significantly lower than the NP69-NC cells（0.405±0.009μg/ml,P0.05）,and the IC50 value of CNE1-d1 cells for DDP（0.671±0.012μg/ml） was significantly higher than that of CNE1-Vector（0.445±0.008/ml,P0.05）.Western blotting results indicated that the fault zone of caspase-3 increased,and Akt phosphorylation at Thr308 and Ser473 decreased in NP69si-Id1 than in NP69-NC,while the fault zone decreased,and Akt phosphorylation at Thr308 and Ser473 increased more significantly in CNE1-Id1 than in CNE1-Vector.Conclusion Id1 can promote the proliferation of nasopharyngeal carcinoma cells and resist the apoptosis induced by DDP,and the mechanism may be related to the up-regulation of p-Akt.