3Δ40p53对p53促肿瘤细胞凋亡作用的影响  

Effect of Δ40p53 isoform on enhancing the pro-apoptotic function of p53 in tumor cells

作　　者：王贵士 赵红伟[2] 乔录新[3] 单军奇 侯庆生[1] 陈德喜[3] 郭洪亮[1] 

机构地区：[1]山东省肿瘤防治研究院山东大学附属山东省肿瘤医院外四科,济南250117 [2]山东省肿瘤防治研究院山东大学附属山东省肿瘤医院病案管理科,济南250117 [3]首都医科大学附属北京佑安医院北京市肝病研究所,100069

出　　处：《中华肿瘤杂志》2017年第5期332-338,共7页Chinese Journal of Oncology

基　　金：山东省自然科学基金（ZR2013HL045）

摘　　要：目的探讨p53亚体Δ40p53对p53促肿瘤细胞凋亡作用的影响。方法体外培养HCT116-p53-/-结肠癌细胞系（内源性表达Δ40p53）、HCT116-p53＋/＋结肠癌细胞系（内源性表达野生型p53）和H1299肺癌细胞系（p53缺失），感染p53腺病毒使p53过表达，转染Δ40p53质粒使Δ40p53过表达。逆转录聚合酶链反应（RT-PCR）检测Δ40p53和p53 mRNA，实时荧光定量PCR检测Δ40p53对p53转录水平的影响，Western blot检测相关蛋白的表达，免疫共沉淀检测Δ40p53与p53相互作用关系，Calcein-AM/PI染色和流式细胞术检测细胞凋亡水平。结果Δ40p53在HCT116-p53-/-细胞中能够稳定转录并表达。实时荧光定量PCR和Western blot检测显示，Δ40p53对p53的转录及表达水平无影响。免疫共沉淀检测显示，Δ40p53与p53能相互结合。Calcein-AM/PI染色显示，H1299-Control组、HCT116-p53-/--Control组、H1299＋p53组、HCT116-p53-/-＋p53组、H1299＋奥沙利铂（Oxa）组、HCT116-p53-/-＋Oxa组、H1299＋p53＋Oxa组和HCT116-p53-/-＋p53＋Oxa组的细胞凋亡率分别为（2.50±0.47）%、（2.40±0.32）%、（5.20±0.58）%、（4.10±0.18）%、（22.40±1.73）%、（19.30±1.11）%、（29.90±1.15）%和（39.30±2.26）%。H1299＋p53＋Oxa组与HCT116-p53-/-＋p53＋Oxa组的细胞凋亡率差异有统计学意义（t＝3.721，P＝0.021）。Calcein-AM/PI染色显示，H1299-Control组、H1299＋Δ40p53组、H1299＋p53组、H1299＋p53＋ Δ40p53组、H1299＋Oxa组、H1299＋Δ40p53＋Oxa组、H1299＋p53＋Oxa组和H1299＋p53＋Δ40p53＋Oxa组的细胞凋亡率分别为（2.60±0.35）%、（2.20±0.17）%、（4.80±0.49）%、（4.90±1.10）%、（20.30±1.10）%、（19.60±1.45）%、（27.90±1.39）%和（35.20±1.43）%，H1299＋p53＋Oxa组与H1299＋p53＋Δ40p53＋Oxa组的细胞凋亡率差异有统计学意义（t＝3.629，P＝0.022）。流式细胞术检测显示，H1299-Control组、H1299＋Δ40p53组、H1299＋p53组、H1299＋p53＋Δ40p53组、H1299＋ObjectiveTo investigate the effect of Δ40p53, an alternative spliced isoform of p53 lacking the N-ter minus, on the pro-apoptotic function of p53.MethodsThe wild-type p53 was ectopically expressed in HCT116-p53-/- （endogenous Δ40p53 expression）, HCT116-p53＋ /＋ （wild-type p53） and H1299 （p53-null） cells by adenoviral delivery, while Δ40p53 plasmid were transfected into these cells to overexpress Δ40p53. The levels of Δ40p53 and p53 mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR） and quantitative PCR. The expression of related proteins was deter mined by Western blotting. The interaction of p53 and Δ40p53 was observed by co-immunoprecipitation assay. Calcein-AM/propidium iodide （PI） staining and flow cytometry were used to detect the apoptotic rate of tested cells in each group.ResultsHCT116-p53-/- cells expressed endogenous Δ40p53 isoform. Neither transcription nor protein expression of wild-type p53 was interfered by the increased expression of Δ40p53. Full length p53 and Δ40p53 could bind to each other. Calcein-AM/PI staining showed that the apoptotic rates of H1299-Control, HCT116-p53-/- -Control, H1299＋ p53, HCT116-p53-/-＋ p53, H1299＋ oxaliplatin （Oxa）, HCT116-p53-/-＋ Oxa, H1299＋ p53＋ Oxa and HCT116-p53-/-＋ p53＋ Oxa groups were （2.50±0.47）%, （2.40±0.32）%, （5.20±0.58）%, （4.10±0.18）%, （22.40±1.73）%, （19.30±1.11）%, （29.90±1.15）% and （39.30±2.26）%, respectively. It was statistically significant between H1299＋ p53＋ Oxa and HCT116-p53-/-＋ p53＋ Oxa groups （t=3.721, P=0.0205）. Moreover, the apoptotic rates of H1299-Control, H1299＋ Δ40p53, H1299＋ p53, H1299＋ p53＋ Δ40p53, H1299＋ Oxa, H1299＋ Δ40p53＋ Oxa, H1299＋ p53＋ Oxa and H1299＋ p53＋ Δ40p53＋ Oxa groups were （2.60±0.35）%, （2.20±0.17）%, （4.80±0.49）%, （4.90±1.10）%, （20.30±1.10）%, （19.60±1.45）%, （27.90±1.39）%, （35.20±1.43）%, respectively. Furthermore, flow cytometry assay

关 键 词：肿瘤 P53 Δ40p53 凋亡 

分 类 号：R734.2[医药卫生—肿瘤]