Human Liver Cancer Metastasis Comparative Proteomics And Systems Biology Research

The research work about this dissertation is referred to many research fields, such as analytical chemistry, chemical biology and clinical oncology. Using different technologies in comparative proteomics and systems biology, we studied to discovery molecular mechanism of hepatocellular carcinoma(HCC) metastasis.The main contributions of this dissertation are as follows:1. Inflammation and anti-apoptosis related pathways were found to play important roles on HCC metastasis.2. Totally 7,034 non-redundant proteins were identified from liver cancer cell lines, the proteomic data was integrated with transcriptomic data in the pilot study of systems biology, indicating that values of TPAE (Transcript-to-Protein Amplification Efficiency) was closely related with subcellular localization of proteins. 3. Low abundant proteins such as SDC4 and ANFPTL4 were detected in extracellular regions in secretome study of HCC cell lines. Metastasis regulator MET had a high level of extracellular expression by proteolysis. What's more, HSPA5 was found to change its subcellular localization among HCC cell lines with different metastatic potentials.4. O-glycosylation and degradation of proteins in metastatic HCC cell lines were studied, indicating that some proteins in lysosome and plasma membrane were actived by degradation.5. In methodology study, different technologies such as 2DE, DIGE, iTRAQ and 1D SDS-PAGE-RPLC-LTQ Obitrap method were found to have distinguish characters on the study of HCC metastatic mechanisms. A novel method was founded in secretome study to be resistant against interference from cell fragments. And a method based on application of magnetic materials to enrich Tryptophan containing peptides was set up and optimized.Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death and occurs highly in Asia and Africa such as China. About 600,000 patients die from HCC world-wide annually. And high metastasis and recurrence remain the major threats to long-term survival. Elucidation of molecular mechanism of HCC metastasis and finding out biomarkers for clinical early and specific diagnose, is very important for improving survival time and life quality of HCC patients. In the work of this dissertation, human HCC cell lines with different metastatic potential but similar gene background were studied. With multiple kinds of proteomic technologies and bio-informatics tools, the mechanism of HCC metastasis was studied in comparative proteomics, systems biology, secretome and post-translational modification proteomics. Totally six parts of this dissertation are discussed as follows:First Part:Background. The improvements of proteomic technology and HCC mechanism study in recent years are summarized in this part. Different proteomic quantification technologies are compared and discussed about their advantages and limitations. Technology development and biological application of sub-cellular proteomics, post-translational modification proteomics, systems biology and bio-informatics were also mentioned. In addition, the construction progress and metastatic characters of the HCC cell lines used in our work are described in this part.Second Part:Comparative proteomics study on HCC cell lines with different metastatic potential. In this part, we studied the comparative proteomic profiles of the four metastatic HCC cell lines for the first time. Instead of traditional single gene reading-out, bio-informatics analysis on the level of biological pathways and protein-protein interaction (PPI) networks were performed. Inflammation and anti-apoptosis related NFκB signaling pathways were enriched in high metastatic HCC cell lines. HDGF, hsp27, HPGD and STMN1 were up-regulated with increase of metastatic potential. Knocking down of HPGD expression with RNAi technology induced apoptosis of HCC cells. Both in the results of 2DE and DIGE, the phenomena of more spots per protein implied that post-translational modifications of proteins are ubiquitous.Third Part:systems biology study on HCC cell lines with different metastatic potential. Using 1D SDS-PAGE-RPLC-LTQ Obitrap, totally 7,034 non-redundant proteins were identified from the four metastatic HCC cell lines and Hep3B cell line. Pathways involved in proliferation were found related with tumorigenesis of HCC through comparative proteomics between these HCC cell lines and normal liver tissues. And anti-apoptosis related pathways were found activated in high metastatic HCC cell lines. Proteomic data was integrated with transcriptomic data. With pilot study of systems biology, no relationship was discovered between the two'omics' data. However, values of TPAE(Transcript-to-Protein Amplification Efficiency)were found to have close relationship with subcellular localizations of proteins. The proteins of low TPAE values were focused in cell nucleolus and extracellular regions while the proteins of high TPAE values mainly occurred in cytoplasm.Fourth Part:Secretome study on HCC cell lines with different metastatic potential. Combined with iTRAQ quantificational technology, a novel method was set up to deal with interference from cell fragments effectively. Low abundant proteins such as SDC4 and ANFPTL4 were detected in the secretome study, and their expressions were found to increase with HCC metastasis. Another metastasis regulator MET was found to express at a high level in extracellular regions by proteolysis. In addition, subcellular localization of HSPA5 was found to change among HCC cell lines with different metastatic potential. The mechanism of localizations and protein functions in HCC metastasis requires further studies.Fifth Part:Post-translational modifications study on HCC cell lines with different metastatic potential. Combined proteolysis and BEMAD methods with DIGE technology, O-glycosyation of proteins in metastatic HCC cell lines were studied. And it was found to be the main reason causing clusters of protein spots on mild alkaline conditions in gel-based experiments. Based on the results of MHCC97L and HCCLM6 with 1D SDS-PAGE-RPLC-LTQ Orbitrap method, degradation profiles of metastatic HCC cell lines were studied using PROTOMAP analytical tool. Some proteins in lysosome and plasma membrane were found to be actived by degradation, such as HEXB, ASAH1 and MET. Their biological functions were focused on proteolysis and cell adhesion. Meanwhile, some enzymes of these degraded proteins were found out in MEROPS enzyme database, such as MMPs and caspase, which play important functions on HCC metastasis.Sixth Part:Application of magnetic materials in enrichment of low abundant proteins in serum. In this part, a novel method was set up based on hydrazide functionalized magnetic microspheres for selective enrichment of Tryptophan (Trp) containing peptides and was applied to enrich Trp-containing peptides from serum for the first time. The content of Trp-containing peptides increased to 80.2% from 19.4% through enrichment. Meanwhile, the complexity of serum sample was reduced greatly and 113 Trp-containing peptides referred to 37 proteins were newly identified.