摘要
目的分析膜联蛋白A7(ANXA7)与肝癌发生的相关性及筛选、鉴定ANXA7的相互作用分子,探讨ANXA7在肝癌发生中的机制。方法通过实时定量PCR法检测48对肝癌与癌旁组织以及多种肝和肝癌细胞株中ANXA7表达量的差异,并通过肝癌细胞ANXA7过表达及特异性干扰抑制分析其对肝癌细胞增殖的影响。采用免疫共沉淀法筛选与ANXA7发生结合的蛋白,并以点突变法分析蛋白质相互作用的关键位点;用蛋白免疫印迹法分析了ANXA7对肝癌相关的重要信号通路中ERK1/2磷酸化水平的影响。结果 ANXA7在肝癌组织与肝癌细胞中均呈下调表达。胰岛素样生长因子结合蛋白2(insulin-like growth factor binding protein 2,IGFBP2)能与ANXA7蛋白发生特异性结合,且IGFBP2上的RGD序列是两者结合的关键位点。肝癌细胞中ANXA7表达上调能抑制肿瘤细胞增殖(P<0.05),并能使IGFBP2介导的ERK1/2的磷酸化水平降低。下调ANXA7的表达可促进肝癌细胞增殖(P<0.01),磷酸化ERK1/2的水平升高。结论 ANXA7可能作为一种抑癌基因,通过介导IGFBP2对ERK1/2磷酸化水平的影响参与对肝癌增殖的调控。
Objective To investigate the role of Annexin A7（ANXA7）in the development of HCC by analyzing ANXA7 expression in hepatocarcinogenesis and identifying its potential interaction molecule.Methods ANXA7 mRNA expression was analyzed by real-time PCR in 48 HCC tissues and tumor adjacent tissues,and different hepatic cancer tissues and cell lines.To analyze the effect of ANXA7 on hepatoma proliferation,ANXA7 was overexpressed or inhibited by specific siRNA in hepatic cancer cells.Co-immunopricipitation（co-IP）method was used to detect the specific binding protein of ANXA7 in HCC cells.The key sites of protein interaction were analyzed by point mutation.Western blotting analysis was used to study the effect of ANXA7 on IGFBP2activated ERK1/2 phosphorylation.Results The expression of ANXA7 was down-regulated in both hepatoma tissue samples and hepatoma cell lines.Insulin-like growth factors binding protein 2（IGFBP2）could specifically bind with ANXA7 through the key RGD site.Up-regulated expression of ANXA7 could inhibit the proliferation of tumor cells（P〈0.05）and decrease the phosphorylation of ERK1/2.Accordingly,down-regulated expression of ANXA7 could enhance the proliferation of tumor cells（P〈0.01）and increase phosphorylation of ERK1/2in HCC cells.Conclusion ANXA7 may serve as a potential tumor suppressive molecule,participating in the regulation of IGFBP2-activated ERK1/2phosphorylation and affecting the proliferation of hepatoma cells.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2015年第4期378-382,共5页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(81071700
31171251)~~
