CBP棕榈酰化位点的突变对皮肤鳞状细胞癌A431细胞生物学行为的影响  

Biological effects of CBP palmitoylation site mutation on cutaneous squamous cell carcinoma A431 cells in vitro

作　　者：高潇 王冰 姚佳 朱玉贞 王静 GAO Xiao;WANG Bing;YAO Jia;ZHU Yuzhen;WANG Jing(Department of Immunology,School of Basic Medical Sciences,Medical College of Qingdao University,Qingdao 266000,Shandong Province,China)

机构地区：[1]青岛大学医学部基础医学院免疫学系,山东青岛266000

出　　处：《中国癌症杂志》2018年第12期906-914,共9页China Oncology

基　　金：青岛市科技计划基础研究项目(KJZD-13-3-2-JCH)

摘　　要：背景与目的:Src羧基末端激酶结合蛋白(Csk-binding protein,CBP)是新近发现的通过棕榈酰化位点锚定脂筏,参与多种肿瘤的发生、发展过程的抑制性跨膜接头蛋白。本研究旨在观察棕榈酰化位点异常的CBP对皮肤鳞状细胞癌A431细胞增殖及凋亡的影响,并探讨其相关的分子机制。方法:构建CBP-增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合蛋白的慢病毒表达载体,采用反转录病毒转染的方法建立CBP棕榈酰化位点突变的A431细胞系以及CBP过表达的A431细胞系,实验分为4组:空白对照组(Parental组,未进行基因转染的A431细胞)、阴性对照组(Control,A431细胞转染仅含EGFP阴性对照病毒载体)、阳性(过表达)对照组(WT-CBP,A431细胞转染WT PAG1-EGFP病毒载体)、实验组(Mutant-CBP,A431细胞转染棕榈酰化位点突变的CBP-EGFP病毒载体)。采用流式细胞术(flow cytometry,FCM)检测转染率,并用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白质印迹法(Westernblot)检测转染后细胞中CBP mRNA表达和蛋白水平,验证转染是否成功。采用流式细胞术检测CBP棕榈酰化位点突变对细胞凋亡的影响;细胞计数试剂盒-8(Cell Counting Kit-8,CCK-8)细胞增殖实验、划痕、transwell迁移及侵袭实验检测CBP棕榈酰化位点突变对A431细胞生长、迁移和侵袭能力的影响;用Western blot检测Csk和Fyn两种下游信号转导分子蛋白水平的变化。结果:建立了稳定的CBP棕榈酰化位点突变A431细胞系和CBP过表达A431细胞系。采用流式细胞术检测细胞凋亡情况,结果显示,与Control组和Parental组比较WT-CBP组A431细胞的凋亡明显增多,差异有统计学意义(P<0.001);但Mutant-CBP组A431细胞的凋亡与WT-CBP组相比明显减少,与Control组和Parental组比较差异无统计学意义(P>0.05)。划痕实验结果表明,与Control组和Parental组比较野生型CBP过表达组A431�Background and purpose:The Csk binding protein(CBP),is palmitoylated and thereby targeted to lipid rafts,which are membrane microdomains characterized by specific lipid and protein compositions,implicated in various aspects of cancer cell biology.This study aimed to investigate the effects of CBP palmitoylation site mutation on proliferation and apoptosis of cutaneous squamous cell A431,and to explore the molecular mechanisms.Methods:The lentiviral vector of CBP-EGFP fusion protein was constructed.The A431 cell line with CBP palmitoylation site mutation and the A431 cell line with WT-CBP were established by retrovirus transfection.The experiments were divided into 4 groups:Parental group(A431 cells without transfection),Control group(A431 cells transfected with only EGFP negative control virus),WT-CBP group(A431 cells transfected with WT-CBP-EGFP virus),Mutant-CBP group(A431 cells transfected with CBP-EGFP virus carrying mutation of palmitoylation sites).CBP mRNA levels were detected by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR),and their protein levels were measured by Western blot.The transfection efficiency was detected by flow cytometry(FCM).After additional culture,the proliferation potential of A431 cells was assayed using Cell Counting Kit(CCK-8),and the apoptotic rate was assessed by FCM.The migration and invasion were assessed by wound healing and transwell assays.The protein levels of Csk and Fyn were detected by Western blot.Results:A431 cell line with stable CBP palmitoylation site mutation or WT-CBP was established.Mutant-CBP inhibited proliferation and induced apoptosis in A431 cell lines.However,the proliferation and induced apoptosis of A431 cells mutated in CBP palmitoylation sites did not change significantly(P>0.05).The WT-CBP significantly decreased the healing rate of A431 cells(P<0.001),while the healing rate of A431 cells mutated in CBP palmitoylation sites did not change significantly(P>0.05).WT-CBP significantly decreased the cell migration and invasion abilit

关 键 词：接头蛋白 CBP 增殖 凋亡 皮肤鳞状细胞癌 

分 类 号：R739.5[医药卫生—肿瘤]