基于Label-free技术的肝癌相关抗原KTN1蛋白质组学研究  

Proteomics study of hepatocellular carcinoma associated antigen KTN1 based on label-free technique

作　　者：潘剑[1] 张瑶尧 覃秋红 陈承晓 黄天明[1] 黄荣师 罗国容[1] Pan Jian;Zhang Yaoyao;Qin Qiuhong;Chen Chengxiao;Huang Tianming;Huang Rongshi;Luo Guorong(School of Basic Medical Sciences,Guangxi Medical University,Nanning 530021,China;Department of Pathology,Guangxi Jiangbin Hospital,Nanning 530021,China;School of Basic Medical Sciences,Guangxi University of Traditional Chinese Medicine,Nanning 530200,China)

机构地区：[1]广西医科大学基础医学院,南宁530021 [2]广西江滨医院病理科,南宁530021 [3]广西中医药大学基础医学院,南宁530200

出　　处：《广西医科大学学报》2022年第7期1073-1079,共7页Journal of Guangxi Medical University

基　　金：广西自然科学基金面上项目(No.2017GXNSFAA198079);广西教育厅项目-广西高校人体发育与疾病研究重点实验室。

摘　　要：目的:鉴定肝癌相关抗原KTN1相互作用蛋白,旨在筛选出与KTN1相结合的重要蛋白,为进一步研究KTN1在肝细胞癌(HCC)中发挥生物学功能的机制提供实验依据。方法:选取HCC细胞株Huh7、HepG2和正常肝细胞株WRL68为实验对象。RIPA法提取各组细胞的总蛋白,考马斯亮蓝染色鉴定蛋白质提取效果,Co-IP实验下拉KTN1蛋白结合蛋白并用Western blotting和蛋白质银染鉴定下拉效果。利用label-free技术对下拉蛋白质进行鉴定,采用ProteinPilot软件分析质谱的结果,采用Skyline软件对蛋白质进行定性和定量分析。利用生物信息学数据库对富集的蛋白质进行GO功能注释和KEGG通路注释分析。结果:考马斯亮蓝染色显示3种细胞的蛋白条带清晰可见。Western blotting和蛋白质银染鉴定Co-IP实验下拉的蛋白中可见KTN1蛋白条带,各组Co-IP富集后均可见10个以上的条带,而阴性对照IgG的泳道则没有蛋白条带或者极少的蛋白条带。Label-free技术共鉴定出29种KTN1结合蛋白,其中H2B1L、IGHG2、VSXL2、RS18、H32、SHRM3、CALL5、SIK3等8种蛋白仅在HCC细胞系Huh7样本中检出。GO分析显示Huh7细胞株中下拉20种KTN1结合蛋白富集于14个生物过程、11个细胞组分和7种分子功能。KEGG通路注释显示这20种蛋白主要富集于Alcoholism和Systemic lupus erythematosus两个通路。结论:在肝细胞癌株Huh7中鉴定出了20种KTN1蛋白结合蛋白,其中H2B1L、IGHG2、VSXL2可能共同参与了KTN1在HCC中的生物学作用。Objective:To identify the interactive protein of hepatocellular carcinoma associated antigen KTN1,and to screen out the important KTN1-binding proteins,so as to provide an experimental basis for further study on the mechanism of KTN1 in the biological function of HCC.Methods:HCC cell lines Huh7,HepG2,and normal liver cell line WRL68 were selected as experimental subjects.The total protein of cells in each group was extracted by the RIPA method,and the effect of protein extraction was examined by Coomassie brilliant blue staining.KNT1-binding proteins were pulled down by Co-IP assay,and the effect of pull-down was detected by Western blotting.and silver staining.Label-free technology was used for identifying the proteins pulled down.Protein-Pilot software was used for analyzing mass spectrometry results,and Skyline software was used for qualitative and quantitative analysis of the proteins.Gene ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway annotation for the enriched proteins were conducted through a bioinformatics database.Results:Coomassie bright blue staining showed clear protein bands of the three cells.Protein bands of KTN1 in the proteins pulled down by Co-IP experiment were identified by Western blotting and protein silver staining.After Co-IP enrichment,more than ten bands were found in each group,while there were no protein bands or very few in the swimming lanes of negative control IgG.A total of 29 KTN1-binding proteins were identified by label-free technique,among which eight proteins including H2B1L,IGHG2,VSXL2,RS18,H32,SHRM3,CALL5,and SIK3 were only detected in Huh7 cell lines.GO analysis showed that 20 KTN1-binding proteins in Huh7 cell lines were enriched in 14 biological processes,11 cell components,and seven molecular functions.Annotations of the KEGG pathway indicated that the 20 proteins mainly concentrated in Alcoholism and Systemic lupus erythematosus pathways.Conclusion:A total of twenty KTN1-binding proteins have been identified in HCC cell line

关 键 词：肝细胞癌 KTN1 免疫共沉淀 相互作用蛋白 

分 类 号：R735.7[医药卫生—肿瘤]