Screening Of Tumor Metastasis Associated Facctors In Sera Of Hepatocellular Carcinoma Patients By Combination Of EMT, PAGE And LC-MS/MS Analyses

In recent years,many potential hepatocellular carcinoma(HCC)-associated serum biomarkers have been identified by proteomic profiling based on mass spectrometry technology.However,the application of these biomarkers in clinical diagnosis and prognosis was limited because of lacking the data of definite biological discription on the biomarker proteins.To overcome these questions,we combined Epithelial-Mesenchymal transit(EMT) examination with Polyacrylamide Gel Electrophoresis(PAGE) fractionation coupled to tandem mass spectrometry(MS/MS) to investigate the dissemination and metastasis related factors in HCC serum proteome.Based on the fact that EMT occurs in dissemination and metastasis of many tumors, we observed the effect of the serum samples from different source on the morphology, cytodynamics,immunocytochemistry of SMMC-7721 human HCC cells and found that serum samples from portal vein of the HCC patients,especially HCC with PVT (portal vein thrombosis),have a remarkable effect on inducing the EMT(epithelial to mesenchymal transition) of SMMC-7721 cells.Then by a novel way,we managed to make a conservative estimate that the EMT-related factors exist in the relevant serum are less than 30 kDa,but more than 3.5 kDa.To further investigate the EMT-related factors in HCC serum proteome,PAGE was used to separate the active serum proteins.During the experiment,we developed a novel quality control technique of separating the serum proteins into five groups and the fourth group of the serum proteins was testified to contain the EMT-related factors. By this way,all high abundant serum proteins and most low abundant proteins disrelated to EMT-induction capability were removed in two steps.Lastly,RP-HPLC(Reversed-phase high performance liquid chromatography) followed by electrospray ion-trap tandem mass analysis was used to examine the fourth group of the serum proteins and many low molecular weight(LMW) proteins under 30 kD were identified. Compared with the traditional way,this experiment offered a significant approach to understand the molecular pathogenesis of HCC.