Differential Proteomic Analysis Of Human Hepatocellular Carcinoma Cell Lines Metastasis Associated Proteins

Differential Proteomic Analysis of Human Hepatocellular Carcinoma Cell Lines Metastasis Associated ProteinsMetastasis and recurrence is the major cause for lower post-operation survival rate of HCC patients. It was indicated that complex aggressive process of metastasis and recurrence were related closely with cancer cell biological behavior (such as adhesion, motility, proliferation), extracellular matrix, liver immunity, tumor angiogenesis, etc). Powerful comparative proteomic techniques with high resolution, high throughput, realtime express analysis can provide effective methods for detecting more key proteins simultaneously during complex pathological progress of multi-gene and multifactor diseases . Unlike traditional ways, more new biomarks and key molecules were found by this strategy to benefit for diagnosis and elucidation of pathological mechanism. Our projects focused on screening and identifying some different proteins between HCC cell lines( MHCC97H and MHCC97L) with high metastatic potential and non- metastasis HCC cell lines (Hep3B) by 2-DE and ESI-MS/MS. We got a group of significant differential proteins including annexin1 and S100A4 etc which may be potential application in diagnosis and prognosis. We also drew a positive conclusion through further analysis and validation of the biological function of annexin1 and S100A4 with anti-sense nucleic acid technique and some experiments related to cell invasion properties . PART 1Comparative proteome analysis of human hepatocellular carcinoma cell lines with different metastasis potentialsThis part 1 has two aspects : (1) To establish a stable and standard comparative proteomic technique platform . (2) Using optimized 2-DE and ESI-MS/MS,comparative study of differential displayed protein expression profiles of hepatocellular carcinoma cell lines with various metastasis potential for screening key molecule related to hepatocellular carcinoma metastasis and recurrence.2-DE is one of critical techniques in comparative proteome analysis, which can separate protein mixtures from each other and show amount and position of different proteins. Stable result from 2-DE protein profile is key point to success of comparative proteome analysis. Through optimizing and comparative analysis, we found some factors including loding mode (cup-loading or in-gel rehydration ),silver staining (diamine silver stains or non- diamine chemical development silver stains, fixation time),cell lysis solution constitute (chaotropes concentration) may play an important role in the quality control of protein expression profile. Separation of total proteins extracted from MHCC97L cell lysate by 2-DE with cup-loading or in-gel rehydration, then the gels were visualized with diamine silver stains or non-diamine chemical development silver stains. Average 1096+40 spots (cup-loading, diamine silver, n=3) and 784+14 spots (in-gel rehydration, non- diamine, n=3) had been detected in protein profile of MHCC97L with Imagemaster( 2D Elite software analysis. Due to more number of protein spots in former and some lower density protein spots in former matched in latter, it indicated diamine silver stains is much better sensitivity than non- diamine chemical development silver stains. Meanwhile sample application by cup-loading can improve separation of proteins in extreme basic area and acid area, better than in-gel rehydration. In addition, we also found high background easily produced with Non- diamine chemical development silver stains.Total proteins of L02 liver cell, which treated with cell lysis buffers containing various chaotropes concentration, were separated by 2-DE .Average spots detected were 925+11 in 7M urea and 2M thiourea extraction, 1019.5+13.5 in lysis buffer containing 9M urea , 781+34 with 8M urea respectively. This result showed various chaotropes concentration could affect distinctly the number of discovered protein spots.Fixation mode of the gel after SDS-PAGE seperation in diamine silver staining was also a keypoint in quilty control. We found maxi...