Quantitative Proteomics Study On The Different Metastatic Ability Of Hepatocellular Carcinoma Tissue And Cells

Hepatocellular carcinoma(HCC) is one of the most life-threatening human cancers in China. Worldwide, it is the fifth-most frequently diagnosed cancer in men, also the second-leading cause of cancer mortality. There are about 700,000 new cases worldwide every year, mainly concentrated in China, Southeast Asia sub-Saharan Africa, where is a significant geographical distribution. HBV is the major cause of HCC and is implicated in the aetiology of as much as 54% of HCC that occurs with HBV. Unfortunately, China has a high incidence of HBV infection. Statistics show that the incidence of infection by the HBV-induced HCC caused by the incidence of male cancer accounted for 7.5 % and 5% mortality. The treatment and prevention of HBV patients is still a public health security problem urgently to China.To date, potentially curative treatments for HCC include hepatectomy, transplantation, or local ablative therapy. While these treatments are promising, the clinical practice has shown that the patients treated for early HCC lesions can have high survival rates and low chances of recurrence and metastasis. Currently Alpha-fetoprotein(AFP) is one of the biomarkers used widely for early diagnosis of HCC. Therefore, deep understanding of the mechanism behind the onset and development of HCC induced by HBV will help to solve these problems.Modern medicine is generally believed that the incidence of cancer is due to the basic mechanisms of cell dysregulate. This imbalance leads to cancer phenotypes occur such as uncontrolled cell division regulation mechanism and value-added uncontrolled cell neoplastic disease and metastasis. Protein quality control mechanisms closely related to tumors phenotype. In cells, the ubiquitin-proteasome pathway play a crucial role in cell proliferation, differentiation, apoptosis, transcription, translation, signal transduction, immune surveillance and other processes through the regulation of the degradation of certain proteins. The development of cancer is a long, multi-stage, multi-process progressive process. The ubiquitin-proteasome system is a multi-factor, multi-step protein degradation control system, the regulation of this system of different factors is an important measure to change the tumor development process. The changes of hepatocellular carcinoma ubiquitin-proteasome pathway is one of the hot points of hepatocellular carcinoma pathology and treatment.In the current study, in order to explore the mechanism of HCC invasion and metastasis, and investigate significant differential or dysregulate pathways, further high coverage, high-accuracy quantitative proteomics technology was applied to study HCC tissue samples and cell lines with different metastatic abilities. To explore the mechanism of HCC invasion and metastasis, and find the significant dysregulated signaling pathways and find the change of protein quality control system, which can enrich the molecular mechanism of HCC.Firstly, we performed a high coverage quantitative proteomics study on the tissues of three distinct stages of tumor-node-metastasis(TNM) using the iTRAQ-based quantitative proteomics approach. A total of 4620 proteins were identified, 3781 of which were quantified. Three hundred and thirty differentially expressed proteins were identified in T1 tumor tissues compared to T1 non-tumor tissues, 132 and 198 proteins were up- and down-regulated, respectively. Three hundred and thirty six proteins were differentially expressed in T2 tumor tissues compared to T1 non-tumor tissues, 172 and 193 proteins were up- and down-regulated, respectively. And 387 proteins were differentially expressed in T3 tumor tissues compared to T1 non-tumor tissues, 185 and 202 proteins were up- and down-regulated, respectively. These differentially expressed proteins were involved in metabolism of exogenous substrate pathway, metabolism of vitamin pathway, cell death pathway, hepatic steatosis, endothelial cancer, proliferation of tumor cell pathway, oxidative stress, cell spreading, cell adhesion and tumor metastasis pathway. It indicated that these changes might be related with HCC development. Subsequent analysis showed that the filamin C(FLNC) protein was significantly up-regulated in the tumor tissue compared with the adjacent non-tumor tissues and the expression level increased gradually with the tumor development. The result has been validated by western blot analysis. This was also confirmed with the RNA-Seq data from 50 independent HCC tumor and adjacent non-tumor tissues in TCGA database. It suggested that FLNC might serve as a new potential biomarker of the HCC development.HCC cell lines with different metastatic abilities, including Hep3 B, MHCC-97 L, MHCC-97 H and HCC-LM6, were selected and combined with the identification method of post-translation modification to investigate tumor cells proteins quality control system, especially the changes and disorders of ubiquitin proteasome pathway. In total, 2,569 ubiquitin proteins were identified. Two hundred proteins were differentially expressed in 97 L compared with Hep3 B, 109 and 91 proteins were upand down- regulated, respectively. Two hundred and fifteen proteins were differentially expressed in 97 H compared with Hep3 B, 109 and 106 proteins were upand down- regulated, respectively. And 97 proteins were differentially expressed in LM6 compared with Hep3 B, 42 and 55 proteins were up- and down- regulated, respectively. These differentially expressed proteins were involved in integrin signaling pathway, actin cytoskeleton signaling pathway, remodeling of epithelial adherens junctions pathway, CDC42 signaling pathway, paxillin signaling pathway, EIF2 signaling pathway, ERK/MAPK signaling pathway and PI3K/AKT signaling pathway and so on.To sum up, based on our high precision and coverage quantitative proteomics technology platform, iTRAQ quantitative proteomics technology and PTM technology, the current study conducted a systematically investigation using HCC tissue samples and cell lines with different metastatic abilities as research model. We had elucidated the changes of the molecular level and signal pathways in liver cancer metastasis process, and established protein profile of HCC cell lines and ubiquitination modification profile of HCC cell lines with different metastatic abilities. We also found the filamin C(FLNC) protein was significantly up-regulated in the tumor tissue compared with the adjacent non-tumor tissues and the expression level increased gradually with the tumor development. It suggested that FLNC might serve as a new potential biomarker of the HCC development and provided targets and insights for diagnosis and treatment of HCC.