S100A4与p53在活细胞内相互作用研究  

Direct interaction of S100A4with p53in the living cells

作　　者：刘臻[1,2] 孟莹 王慧君 Philip S.Rudland Roger Barraclough 张述 LIU Zhen;MENG Ying;WANG Hui-jun;Philip SRudland;Roger Barraclough;ZHANG Shu(School of Medicine and Life Sciences,University of Jinan-Shandong Academy of Medical Sciences,Jinan 250022,P.R.China;Department of Gastroenterology Oncology,Shandong Cancer Hospital and Institute,Shandong First Medical University and Shandong Academy of Medical Sciences,Jinan 250117,P.R.China;School of Biological Sciences,University of Liverpool,Liverpool L693BX,U.K)

机构地区：[1]济南大学·山东省医学科学院医学与生命科学学院,山东济南250022 [2]山东省肿瘤防治研究院(山东省肿瘤医院)消化肿瘤内一科,山东第一医科大学(山东省医学科学院),山东济南250117 [3]利物浦大学生物科学学院,利物浦英国L693BX

出　　处：《中华肿瘤防治杂志》2020年第6期431-437,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：山东省医药卫生科技发展计划(2017WS266);山东省医学科学院科研项目(201730,201838)。

摘　　要：目的体外研究发现S100A4蛋白可与p53蛋白结合,但至今尚不清楚S100A4与p53蛋白能否在活细胞内发生相互作用。本研究旨在确定S100A4与p53在活细胞内的相互作用。方法采用光学生物传感器体外检测S100A4蛋白与p53蛋白的结合能力。应用激光共聚焦显微镜的荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术检测Hela细胞(人宫颈癌细胞株)内S100A4与p53蛋白的相互作用。结果光学生物传感器体外检测发现,野生型S100A4蛋白与p53蛋白结合能力强,对照组突变型S100A4蛋白则失去与p53蛋白的结合能力。采用FRET技术发现,表达CFP-S100A4和p53-YFP融合蛋白的Hela细胞中,细胞核内p53-YFP的荧光强度衰减后,CFP-S100A4的荧光强度增强,发生FRET;而细胞质中没有发生FRET。作为对照,表达突变CFP-mS100A4-C和p53-YFP融合蛋白,或表达S100A4和YFP的Hela细胞内没有发生FRET,表明S100A4和p53之间的相互作用发生在活细胞的细胞核中。结论活细胞内S100A4可直接与细胞核中的p53相互作用,这可能是S100A4蛋白在体内发挥生物学作用的分子基础,提示阻断S100A4的钙结合位点可能是潜在的抗肿瘤转移作用靶点。OBJECTIVE To determine the interaction of S100A4 with p53 and to find the location of S100A4 interacting with p53 in living cells.METHODS Using an optical biosensor to examine the ability of S100A4 binding to p53 in vitrofirstly,and then to detect S100A4 interacting with p53 in living Hela cells(Human cervical cancer cell line)using the fluorescence resonance energy transfer(FRET)technique with laser con-focal microscopy.RESULTS Wild-type S100A4 protein had a strong binding ability with p53 protein in vitro,while mutant S100A4 protein in the control group lost the binding ability with p53 protein.Imaging FRET in Hela cells expressing CFP-S100A4 and p53-YFP showed an increase in CFP fluorescence intensity in cell nuclei,after the photobleaching of YFP in cell nuclei.Comparing to cell nuclei expressing S100A4 and YFP,the cell nuclei expressing mutants S100A4-C(CFP-mS100A4-C)and p53-YFP and expressing S100A4 and YFP showed no occurrence of FRET.This demonstrated that the interaction between S100A4 and p53 occurred in the nuclei of living Hela cells.However,FRET did not occur in the cytoplasm of cells,showing that S100A4 did not interact with p53 in the cytoplasm.CONCLUSIONS S100A4 can interact directly with p53 in the nuclei of living cells.This might be a molecular basis for S100A4 inducing tumor metastasis in vivo.The results also suppose that the inhibition of the calcium-binding site of S100A4 might be a possible anticancer target.

关 键 词：荧光共振能量转移 相互作用 肿瘤转移 P53 S100A4 

分 类 号：R73-3[医药卫生—肿瘤]