Discovery And Preliminary Verification Of Hepatocellular Carcinoma Metastasis-associated Secreted Proteins

Hepatocellular carcinoma (HCC) is the seventh common cancers worldwide. The mortality of HCC is very high, only behind breast cancer and lung cancer. About 383,000 people die from HCC in China every year, accounting for 50% of liver cancer deaths in the world. Metastasis is the main factor leading to treatment failure and high fatality of HCC However, the molecular mechanism of hepatocellular carcinoma metastasis is not clearly understood. Effecient biomarker or biochemical test indicating hepatocellular carcinoma recurrence has not been found. It is ugent to discover reliable prediction markers for HCC metastasis.The development of tumor metastasis depends on the mutual communication between cancer cells and their stromal milieu. The molecular interactions of metastatic tumor cells with host cells and extracel-lular matrix (ECM) enable the tumor cells to modify the stromal components and turn them into accomplices for ECM remodeling, immune suppression, host tissue destruction and angiogenesis, all of which are essential for metastasis. It is generally conceived that tumor-derived secreted factors play critical roles in this abetting process. Secreted proteins can not only regulate development of tumor by autocrine or paracrine, but also be secreted into the blood, urine, interstitial fluid and serve as clinical markers for tumor metastasis. Proteomics is a global, high-throughput protein analysis strategy. It is widely used to study the pathogenesis of complex, multigenic and multifactorial diseases including cancer. We used stable isotope labeling with amino acids in cell culture (SILAC), a quantitative proteomics technique, to analyze secretory proteins of HCC, in order to find HCC metastasis-associated proteins.Three HCC cell lines (MHCC97L, MHCC97H and HCCLM6) with the same genetic background and gradually increased metastatic potentials were used for SILAC analysis of secretory proteins extracted from serum-free medium. Totally,1287 proteins were identified, of which 18% are secretory proteins, cell membrane and cell junction proteins. There were 890 proteins identified in MHCC97H/MHCC97L, of which 174 were differentially expressed proteins (ratio>±1.5 fold), including 40 up-regulated proteins and 134 down-regulated proteins in MHCC97H. At the same time,983 proteins were identified in HCCLM6/MHCC97L, of which 182 were differentially expressed proteins, including 67 up-regulated proteins and 115 down-regulated proteins in HCCLM6. Biology process analysis of upregulated proteins discovered that cell adhesion, regulation of gene expression, nucleic acid metabolic process, protein metabolic process, carbohydrate metabolic process, cell differentiation, DNA metabolic process, cell recognition, biopolymer modification and lipid metabolic process were significantly enriched. Pathway analysis of upregulated preoteins showed that lysosome, other glycan degradation, ECM-receptor interaction and sphingolipid metabolism were significantly enrichiched. Fourteen differentially expressed proteins gradually increased with the increasement of metastatic potentials of cell lines, including 6 proteins have positive correlations with tumor metastasis (such as MMP1、ANXA1、FGL1), which reflect the reliability of our data. We identified four novel proteins (COL6A1, LUM, GNS, PCSK9) without previous report about the relationship with HCC metastasis.We validated the expression level of COL6A1 (MHCC97H/MHCC97L=2.77, HCCLM6/MHCC97L=4.23) by Western Blot in conditioned media from six cell lines (LO2, SK-Hepl, HepG2, MHCC97L, MHCC97H and HCCLM6). LO2 is a kind of normal liver cell line, SK-Hepl and HepG2 are HCC cell lines without metastatic ability. COL6A1 could not be detected in either conditioned media or whole cell lysate from normal liver cell line and non-metastatic HCC cell lines. The expression level of COL6A1 was gradually incressed in secretomes of MHCC97L, MHCC97H and HCCLM6 but gradually decreased in the whole cell lysate. This result indicated that the secreted amount of COL6A1 was positively associated with the metastastic potentials of HCC cells.COL6A1 commonly exists in extracellular matrix, mediating microfiber network and basement membrane formation. COL6A1 may be involved in changing the characteristics of ECM and regulating cell movement. These functions may promote metastasis of HCC. In this research we preliminarily verified that secreted COL6A1 is positively correlated with metastasis of HCC, but the concrete mechanism needs to be further studied.We also tried to detect COL6A1 in the sera from HCC patients with different metastatic conditions by Western Blot. However, it could not be detected in serum directly due to the very low aboundance. We were also working on the assembling of ELISA kit for detection of COL6A1 in serum samples.Quantitative proteomics analysis was also performed on whole cell lysates of HCC cell lines MHCC97H and HCCLM6. Using iTRAQ4plex labeling, a total of 4804 proteins were identified and 4790 of which were quantified. There were 103 differentially expressed proteins in HCCLM6 cell line compared with MHCC97H (ratio ≥±1.5), among which 46 proteins were up-regulated and 57 proteins were down-regulated in HCCLM6. Most of the differentially expressed proteins were located in cytoplasm and cell membrane. GO analysis suggested that proteins related to cell adhesion and protein binding were significantly enriched. This work discovered four novel candidate HCC metastasis-associated proteins (Laminin subunit alpha-5, Tubulin beta-2B chain, Centromere protein F, Vesicle-associated membrane protein 5), providing a valuable reference for future studies on HCC metastasis.