摘要目的 筛选及验证肝癌细胞中与转录激活因子3(ATF3)相互结合的蛋白质,初步探讨在肝癌发生发展中协助ATF3发挥生物学功能的相关分子机制.方法 应用免疫沉淀法沉淀出HepG2细胞中与ATF3相互结合的蛋白,经变性聚丙烯凝胶分析,与空载体组比对,挑选出差异性的电泳条带进行蛋白质谱分析,筛选出候选蛋白;再经免疫共沉淀验证二者是否相互结合;应用免疫组织化学及Western blot检测并分析ATF3和候选蛋白在肝癌中的表达模式.结果 过表达ATF3的HepG2实验组呈现出差异性表达的电泳条带;经质谱检测,Mascot软件分析及NCBI数据库检索,得到候选蛋白肽段信息,结合前期实验结果,PubMed文献报道,从中筛选出与ATF3最可能相关的蛋白gelsolin(GSN);免疫共沉淀结果验证了ATF3与GSN相互结合的关系,免疫组织化学及Western blot检测出肝癌组织中ATF3与GSN的表达模式一致.结论 ATF3可能通过与GSN蛋白之间的相互作用,在肝癌形成过程中协同发挥抑癌作用.

abstractsObjective To identify and verify proteins that interact and collaborate with ATF3 in inhibiting hepatocarcinogenesis.Methods Immunoprecipitation (IP),co-IP and protein spectrum analysis were used to identify the protein which interacted with ATF3 in HepG2.Immunohistochemistry (IHC) and Western blot (WB) were used to detect the expression pattern of ATF3 and its candidate interacting proteins in liver tissue.Results The protein expression differences were detected by IP in two HepG2 groups.The experimental group was infected by lentiviral vector with ATF3 over-expression and the control group was infected by mock-vehicle.Several protein bands with expression diversity were analyzed by protein spectrum,which revealed several candidate proteins that may be related with ATF3.Peptide sequences were analyzed by Mascot software and NCBI database.Combined with the existing literature and our study results,Gelsolin (GSN) was identified as a protein closely interacting with ATF3 and confirmed by co-IP,IHC and WB.Conclusions GSN is identified and verified as an interacting protein with ATF3.ATF3 may function as a suppressor of liver cancer via protein-protein interactions with Gelsolin.