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Int J Mol Sci. 2015 Feb 16;16(2):4209-25. doi: 10.3390/ijms16024209.

Phosphoproteomic analysis of the highly-metastatic hepatocellular carcinoma cell line, MHCC97-H.

Tian M1, Cheng H2, Wang Z3,4, Su N5, Liu Z6, Sun C7, Zhen B8, Hong X9, Xue Y10, Xu P11,12.

Author information

1
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China. mmtian@genetics.ac.cn.
2
Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. chenghan@hust.edu.cn.
3
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China. wangzq@whu.edu.cn.
4
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430072, China. wangzq@whu.edu.cn.
5
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China. sunamask@gmail.com.
6
Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. lzx@hust.edu.cn.
7
Tianjin Baodi Hospital, Tianjin 301800, China. husband88@163.com.
8
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China. zp1963@sina.com.
9
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430072, China. xhy78@whu.edu.cn.
10
Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. xueyu@hust.edu.cn.
11
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing 102206, China. xupingghy@gmail.com.
12
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430072, China. xupingghy@gmail.com.

Abstract

Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC-MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites) of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO) categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2), Eukaryotic Initiation Factor 4A-III (EIF4A3), Cell Division Cycle 5-Like (CDC5L) and Survival Motor Neuron Domain Containing 1 (SMNDC1), were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing.

PMID:
25690035
PMCID:
PMC4346953
DOI:
10.3390/ijms16024209
[Indexed for MEDLINE]
Free PMC Article

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