Quantitive Membrane Proteomic Study On The Mechanism Of Migration Of Hepatocellular Carcinoma

Wordly, over 55 % of the reported primary liver cancer and over 300,000 death were casused every year in China. Among of which, over 90 % of the primary liver cancer patients is hepatocellular carcinoma(HCC). The main properities of HCC including high malignant degree, easy to migration, and short prognosis survival time. The clinical diagnose of HCC is mainly based on the detection of AFP expression level and medical image technologies such as B ultrasonic waves. But the correnlation of AFP positive to HCC is only 70-80 % while B ultrasonic waves can not diagnose of HCC with small volume at the very beganing stage. Surgical remoal of HCC tisue is still the first choice though the easy migration properity make the poor prognosis after surgical. Thus, the understanding of HCC mechanism and discovery of diagnose or therapeutic markers is thirsted for the HCC preventiation, diagnose and prolong prognosis.One of the widely accepted molecular mechanisms of HCC migration including micro-environment change caused hypoxia and even further epithelial-mesenchymal transition(EMT). during this process, the ECM and plasma membrane changed and then the carcinoma cell acquired the invision and migration properity. Especially during HCC, the liver cell damage caused by virus affection and micro-environment change make some of the hepatocyte cells acquired stem-cell-like properity(progenitor cell). These progenitor cells played important roles in HCC development and migration. The deep research of the ECM and plasma membrane change durning EMT is very improtatnt in understanding the mechanism of HCC disease.In this study, a whole membrane proteomic process was optimized and setup based on HepaRG and MCF7 cell lines, firstly. The ultra-centrifugation based and two-phase separation based membrane enrichment methods were first optimized and comparied systamitically. The whole membrane protein enrichement method can acquire membrane protein fraction with high purity(over 40 %, number based). Based on the different enzyme digestion methods including in-gel digestion, in solution digestion with detergent and enhanced FASP methods: 1) over 1000 membrane proteins can be idenditified in the optimized methods; 2) the in solution digestion with RapiGest(ISD-RG) combined with high pH RPLC identified more membrane proteins while without bias of in gel digestion. To our knowledge, the membrane protein dataset acquired in this research is the largest membrane protein dataset based on single cell lines(Rui, 2013, 486 membrane proteins; Hu, 2011, 495 membrane proteins).In the second part, so as to make a full understanding of the function of progenitor cell in HCC development and migration, HepaRG cell line was cultured and induced, then enriched the membrane proteins of non-defferentiation and differentiation stages. The enriched membrane proteins of different stages were quantitatively analyzed by iTRAQ proteomic. The IPA analysis proved the 70 different expressed membrane proteins of different stages were enriched in ECM composion, the enriched bio-function including cell adhersion, migration, and cancer besides the metabolism function acquired after differentiation. Several key proteins such as MMP14, HYOU1, CD44 and OCLN were further evaluated. An even further evaluation of MMP14 in HCC cell lines and tissue samples proved the correlation of MMP14 with HepaRG and HCC migration. The understanding of the expression profile of membrane proteins especially the ECM related proteins during HepaRG differentiation is helpful to probe the progenitor cell’s function during HCC.In the last part, hepatocellular carcinoma cell lines, MHCC97L(low migration properity) and HCCLM6(high migration properity) were systematically evaluated by quantitive membrane proteomic and whole transcriptome sequencing strategries. Finally 96 membrane proteins were up-regulated while 48 proteins were down regulated in LM6 cell based on membrane proteomic analysis. In the transcriptome sequencing part, among the 16,558 qualified protein-encoding genes, 1,472 protein-encoding genes were statistically differently expressed, based on IPA analysis, the migration differentiation of the two cell lines including signal pathways such as TGF-β, VEGF, IL6, EGFR, TNF-α, and FGF, these singal pathways also connected by the degradation of ECM composion proteins such as colleagen, cadherin and intergrin by extracellar matrix metalloproteinases such as MMP14. The TCGA analysis of the differently expressed proteins/genes of quantitive membrane proteomic and transcriptome sequencing showed 29 genes were differentially expressed between tumorous part(TP)and non-tumorous part(NT)(p<0.001), especially 12 genes were statistically differently expressed((p<0.0001)). The expression level of potential target genes such as ATR, CHST11, FMO3, MOCS2, NUCB2 and ST6GALNAC4 are correlated to the total survival rate of HCC patients. These research may shed light on the mechanism of metastasis on HCC.