Cell Surface Glycan Profiling In Epithelial Mesenchymal Transition Process Of Huh7Hepatocellular Carcinoma Cell Induced By HGF

Epithelial mesenchymal transition (EMT), a critical step of tumormetastasis, has become a hot spot in the research of tumor metastasis.Metastasis is the major obstacle of improvement of HCC treatment efficacy andsurvival. Therefore, for the screening and validation of metastasis-relatedbiomarkers and interventional targets are the main emphasis and urgentassignment on the field of HCC research.Most of the tumor biomarkers are glycoprotein. More and more studiespaid their attention to the HCC glycomics research based on the abnormalglycosylation and glyco-markers. The goal of this study was to identify HCCmetastasis related differential glycan pattern and glycan processing enzyme; weestablished the HGF induced HCC EMT model in vitro. Lectin microarray wasused to determine the differential cell surface glycan profiling. Furthermore, wealso explored the enzymatic basis of the dreamatic change of glycan byqRT-PCR. It may provide novel insight for forecasting HCC metastasis, earlydiagnosis, establishing glycan-related molecular targets and improving theprognosis. PART ONE CREATION AND VALIDATION OF HCC HUH7CELLEMT MODELIt has reported that EMT played pivotal roles in tumor invasion andmetastasis. The aim of this part was to establish the HGF-induced EMT model.We used10ng/mL HGF to stimulate HCC Huh7cell. Cell morphology wasobserved by inverted phase contrast microscope. The expression of EMT-relatedmarkers was determined by qRT-PCR and western blot analysis. Transwellchamber invasion and cell adhesion assay were used to evaluate the change ofcell invasion and adhesion ability. After HGF treatment, Huh7cellularmorphology was converted to a diffused fibroblast-like morphology. For thebiomarker of EMT, the data demonstrated the down-regulation of E-cadherin(epithelial marker) and the up-regulation of N-cadherin, vimentin and α-SMA,snail, slug and twist (mesenchymal markers). And it is more obvious as with thegrowth of induction time. In addition, the stimulation of HGF can enhance theinvasive ability and weaken the adhesion ability. These changes demonstratedthat HGF could induce a typical EMT model.PART TWO CELL SURFACE GLYCAN PROFILING ANDENZYMATIC BASIS OF HCC EMT-RELATED MODELGlycosylation is the most common post-translational modificationaccompanying with the development, invasion and metastasis of many cancer. Inthis part of study, we used lectin microarray to detect the differential profiling ofcell surface glycan. The difference was validated by lectin blot and fluorescencecell lectin-immunochemistry. Finally, qRT-PCR was used to investigate theenzymatic basis of glycan. The results demonstrated a decreased affinity inseven lectins: ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan ofcell surface glycoproteins for the HGF-treated cells. This implied that glycan containing T/Tn-antigen, NA2and bisecting GlcNAc, Sia α2-6Gal/GalNAc,terminal α or β GalNAc structures were reduced. While the binding ability ofthirteen lectins: AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL,ECL, GSL II and PHA-L to glycan were elevated, and a definite indication thatglycan containing α Fuc and Sia-Le, core fucose, α-man, α (β)-GlcNAc, β1,6GlcNAc branching and tetraantennary complex oligosaccharides structures wereincreased in HCC EMT cells. These results were further validated by lectin blotand fluorescence cell lectin-immunochemistry. Furthermore, the mRNAexpression level of MGAT3decreased while that of MGAT5, FUT8and β3GalT5increased in HCC EMT cells. These finding mentioned above indicated that thecell surface glycan pattern had dreamy changed in the HCC EMT process and itmay coincide with the expression of glycosyltransferase. Also it showed that thealterations of related glycosyltransferases were closely related to HCCmetastasis.CONCLUSIONS1. We successfully established the HGF-induced Huh7HCC cell EMT model.2. Result from differential cell surface glycan profiling indicated that the cellsurface glycan such as T/Tn-antigen, NA2and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or β GalNAc structures were decreased, and theterminal α Fuc and Sia-Le, core fucose, α-man, α (β)-GlcNAc, β1,6GlcNAc branching structures and tetra-antennary complex oligosaccharidesstructures were increased in the EMT process of Huh7cells.3. For the enzymetic basis of the change of cell surface glycan, the mRNA expression level of MGAT3decreased while that of MGAT5, FUT8andβ3GalT5increased in HCC EMT Huh7cells.