title: Dynamic expression patterns of differential proteins during early invasion of hepatocellular carcinoma.
abstract: BACKGROUND
  Tumor cell invasion into the surrounding matrix has been well documented as an early event of metastasis occurrence. However, the dynamic expression patterns of proteins during early invasion of hepatocellular carcinoma (HCC) are largely unknown. Using a three-dimensional HCC invasion culture model established previously, we investigated the dynamic expression patterns of identified proteins during early invasion of HCC.
  
  
  METHODS
  Highly metastatic MHCC97H cells and a liver tissue fragment were long-term co-cultured in a rotating wall vessel (RWV) bioreactor to simulate different pathological states of HCC invasion. The established spherical co-cultures were collected on days 0, 5, 10, and 15 for dynamic expression pattern analysis. Significantly different proteins among spheroids at different time points were screened and identified using quantitative proteomics of iTRAQ labeling coupled with LC-MS/MS. Dynamic expression patterns of differential proteins were further categorized by K-means clustering. The expression modes of several differentially expressed proteins were confirmed by Western blot and qRT-PCR.
  
  
  RESULTS
  Time course analysis of invasion/metastasis gene expressions (MMP2, MMP7, MMP9, CD44, SPP1, CXCR4, CXCL12, and CDH1) showed remarkable, dynamic alterations during the invasion process of HCC. A total of 1,028 proteins were identified in spherical co-cultures collected at different time points by quantitative proteomics. Among these proteins, 529 common differential proteins related to HCC invasion were clustered into 25 types of expression patterns. Some proteins displayed significant dynamic alterations during the early invasion process of HCC, such as upregulation at the early invasion stage and downregulation at the late invasion stage (e.g., MAPRE1, PHB2, cathepsin D, etc.) or continuous upregulation during the entire invasion process (e.g., vitronectin, Met, clusterin, ICAM1, GSN, etc.).
  
  
  CONCLUSIONS
  Dynamic expression patterns of candidate proteins during the early invasion process of HCC facilitate the discovery of new molecular targets for early intervention to prevent HCC invasion and metastasis.
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Rong-Xin
authors.initials: RX
authors.lastname: Chen
authors.affiliation: Institute of Digestive Disease, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Song
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Yin-Ying
authors.initials: YY
authors.lastname: Dong
authors.affiliation: Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, PR China.
authors.forename: Chao
authors.initials: C
authors.lastname: Hu
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Qiong-Dan
authors.initials: QD
authors.lastname: Zheng
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Tong-Chun
authors.initials: TC
authors.lastname: Xue
authors.affiliation: Institute of Biomedical Science, Fudan University, Shanghai, PR China.
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Liu
authors.affiliation: Institute of Biomedical Science, Fudan University, Shanghai, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Zheng-Gang
authors.initials: ZG
authors.lastname: Ren
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Yin-Kun
authors.initials: YK
authors.lastname: Liu
authors.affiliation: Liver Cancer Institute, Zhongshan Hospital, Fudan University & Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, PR China.
authors.forename: Jie-Feng
authors.initials: JF
authors.lastname: Cui
mesh_terms: D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D015972:Gene Expression Regulation, Neoplastic; D052138:Genes, Neoplasm; D006801:Humans; D007553:Isotope Labeling; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000091402:Prohibitins; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
chemical_list: D009363:Neoplasm Proteins; C000717331:PHB2 protein, human; D000091402:Prohibitins