Identification Lymphatic Metastasis-associated Proteins In Mouse Hepatocarcinoma Cell Lines Using Quantitative Proteomics Technique

Background: Invision and metastasis are the fundamental characters in differentiating the malignant tumer from benign one,and are the major causes of refractoriness of tumer . High mortality ,poor prognosis and uncertain pathogenesis deriving from metastasis of malignant tumor are still one of tough problems in tumor research for prolonged time.Because majority of malignant tumors are carcinomas and lymph node metastases often represent the first step in the metastatic process,it is very important to make the molecular mechanisms of lymphatic metastasis clearly,and to construct a set of efficient repression methods.Hca-F and Hca-P are a pair of syngenetic mouse hepatocarcinoma ascites cell lines ,presenting a specific potential of lymphogenetic metastasis when inoculated subcutaneo- usly in 615 mice. Hca-F with a highly metastastic potential (>70%)and Hca-P with a low one( < 30%)are rare and available cell model as a reference each other in the research of the molecular mechanisms of lymphatic metastasis.We have investigated the gene expression of Hca-F and Hca-P in mRNA level using cDNA microarray,and have found the conspicuous differential gene expression. However,studying in mRNA level only represents the transcriptive regulation,and can't reflect the abundance ,the complicated post-translation modification,location and transition in subcellar and the interaction of proteins etc.Protein is a executant of physiological function and a direct performer of life.The mechanisms of physiology and pathology will be elucidated by investigating protein.Proteom is to study all the proteins in a cell or in a tissue.It is difficult to sepratate and to abtain all the expressed proteinsowing to the restriction of the modern technique.So developing high-through technical methods will facilitate proteomics.To get the destination, two-dimensional gel electrophoresis(2DE) and mass spetromery (MS)as the basic techniques of sepratation and identification have been developed.Object: In order to obtain lymphatic metastasis-associated proteins, the protein expressed profiles of mouse hepatocarcinoma ascites syngeneic cell lines Hca-F (highly metastatic)and Hca-P(low metastatic) were compared using fluorescent two-dimensional difference gel electrophor- esis(2D DIGE)-quantitative proteomics which has been developed recently,and then differential protein spots with statistical significant between Hca-F and Hca-P were identified by mass spetromery (MS).Method: The anabiosis cells of Hca-F and Hca-P were intraperiton- eally injected to culture 2 passage in 615 mice,and cultured in vitro for 24 hours.Collected tumor cell,which of half part for the animal experiment to detect the metastaic rate of lymphtic nodes,another half one for extracting total protein. Hca-F and Hca-P were injected subcutaneously in the hypoderma of right axillary line to observe 28 days ,and executed 20 mice. All draining lymphtic nodes were exsected to be stained with HE . The metastaic rate of lymphtic nodes were calculated.The tumor cells were washed and were centrifuged in a refrigerated centrifuge. The deposition was transfered into a reaction tube containing lysis buffer Lysates to be sonificated,and were centrifuged in a refrigerated centrifuge. Supernatant was total protein,and protein concentration of lysates were determined by Bradford assays. Labeling reactions using CyDyes were performed following the manufacturer's instructions. For the preparation of the"internal pooled standard,"equal protein amounts of the two corresponding twins were pooled and labeled with Cy2. Equal protein amounts of Cy2, Cy3, andCy5 labeled samples were mixed and added to an equal volume sample buffer and rehydration buffer .DryStrips were rehydrated for 10 hours in rehydration buffer. IEF was performed using IPGphⅡ.After anodic cup loading, the IPG strips were focused for a total of 76 kVh at 50mA. Prior to electrophoresis each strip was equilibrated in equilibration buffer . 2-DE was performed using a stacking gel and a separation gel. The gels werescanned with a Typhoon 9400 using the parameters suggested by the manufacturer.Image analysis was performed with DeCyder. The differential protein spots with statistical significant were excised by an automated spot excitation robot, recovered the spots in a 96-well plate. Protein identification was performed by an in-gel digestion method. Mass spectrometric analysis of tryptic digests was performed usingLC- ESl- MS/MS. Sequest software researched NCBInr database to identify proteins. The identified proteins were verified by Western blotting.All the identified proteins were analized by DAVID and PANTHER at cell component, molecular function ,biological process and metabolism and pathway.Result: The rates of tumor formation of Hca-F and Hca-P cells were both 100%,and the metastatic rates of lymphtic nodes were 75% and 25% respectively,with statistical significance(p<0.05). Hca-F and Hca-P cell lines consistented with the requirement of the experiment.The differential expression of protein profile of Hca-F and Hca-P have been established.DIA mode of DeCyder may detect three overlapping images in one gel ,labeled with Cy2, Cy3 and Cy5 respectively ,and may abtain precise radio. BVA mode of DeCyder may match among the gels ,and may detect the diference which can be calculated level of confidence by statistic in all the gels . 163 differential protein spots with statistical significance between Hca-F and Hca-P were detected, including 23 differential spots of more than 2 fold; 79 of 1.5 fold to 2 fold; 61 of 1 fold to 1.5 fold. 86 protein sprots were up-regulated in Hca-F and 77 protein spots were down-regulated in Hca-F. All of the protein differential spots were digested and analyzed using MS,and 168 proteins were identified by NCBInr database using sequest software.The differential expression proteins more than 2 fold : Vcp, Eef2, Tkt, Pkm2 ,Gls,Ero1, Aldh2, 1810047C23Rik, Ruvbl1, Eef1α2, Vim, Prph, Des, Anxa7, Ckb, Mdh2, Anxa5, Ech1, Hnrpa2b1, Lactb2, GSTο1, UCHL3, ERp29,Crk, Lypla1, Stmn1. The identified proteins were verified by Western blotting. ERp29 and UCHL3 were down-regulated in Hca-F ,as same as the result of identification.The analysis of bioinformatics of 168 proteins revealed that the identified proteins were principally located in intracellular organelle,incluing mitochondrion, cytoskeleton, and endoplas- mic reticulum etc.The identified proteins are involved chaperone ,cytoskeletal protein , hydrolase , nucleic acid binding , oxidoreductase , protease , select calcium binding protein , select regulatory molecule , transcription factor , transfer/carrier protein ,transferase etc.The analysis of biology process revealed that the identified proteins mainly participate in protein metabolism and modification, nucleoside, nucleotide and nucleic acid metabolism, cell structure and motility, immunity and defense etc. The analysis of metabolism and pathway revealed that the identified proteins mainly participat in arginine and praline metabolism,proteasome, glycolysis/gluconeogenesis,carbon fixation,cell communication,regulation of actin cytoskeleton,focal adhension, antigen processing and presentation etc.Conclusions: In order to obtain lymphatic metastasis-associated proteins, the protein expressed profiles of mouse hepatocarcinoma ascites syngeneic cell lines Hca-F (highly metastatic)and Hca-P(low metastatic) were compared using 2D DIGE,followed by MS to identify the differential protein spots with statistical significant. High-through quantitative proteomics technique is a powerful tool to identify lymphtic metastasis- associated proteins. 163 differential protein spots in 2D DIGE were detected, including 86 protein spots up-regulated in Hca-F and 77 protein spots down-regulated in Hca-F. Followed by MS and researching in database,168 proteins were identified. The analysis of bioinformatics of the identified proteins revealed that they were principally located in intracellular organelle,especially in mitochondrion; nucleic acid binding , oxidoreduc- tase and cytoskeletal protein accounts majority of identified proteins in molecular function ; the identified proteins mainly participate in protein metabolism and modification, nucleoside, nucleotide and nucleic acid metabolism, cell structure and motility, immunity and defense etc.; regulation of actin cytoskeleton ,focal adhension and antigen processing and presentation etc. perhaps were the key pathways in lymphtic metastasis of tumor.