| Background:Hepatocellular carcinoma(HCC)is a common malignant cancer of the liver,patients have a low five-year survival rate after clinical diagnosis.The main cause of such poor prognosis and high mortality is the limited treatment options,and its recurrence and metastasis of HCC.It is well-known that glycosylation plays an important role in cancer occurrence and metastasis,which not only participates in regulating the growth and proliferation of cancer cells,but also promotes cancer metastasis and invasion through affecting the adhesion between cancer cells.However,the molecular mechanism of glycosylation involved in the development of hepatocellular carcinoma remains unclear.In this study,HCC tumors with different clinical stages and epithelial-mesenchymal transition cell models were used for identifying and quantifying the dynamic changes of N-linked glycosylation in the development of hepatocellular carcinoma.This study not only revealed the dynamic changes of N-linked glycosylation in the development of HCC,but also clarified the molecular mechanism of HCC-related glycoprotein to promote the EMT progress of HCC cells.Methods:In this study,A total of 17 samples were collected and used for proteomic and glycoproteomic analysis;The data of peptides and glycopeptides were searched and quantified to discover the altered glycoproteins,and the relevant bioinformatics analysis were further performed.In addition,three HCC cell lines were treated by HGF and TGF-?1to induce EMT,respectively.Then the HGF/TGF-?1-induced EMT were further evaluated by the m RNA and protein levels of EMT-related markers.Their whole cellular proteins were then harvested at different time points for the labeled proteomics and glycoproteomics analysis.Finally,the gene-knockdown and overexpression technology were used to explore the possible mechanism of altered core-fucosylation during the EMT process.And five mutated cell lines of FOLR1 gene were constructed to determine the function of FOLR1 in EMT process.Results:We totally identified 3,139 unique N-linked intact glycopeptides from all HCC samples.By relatively quantifying the intact glycopeptides among three sample groups,we totally identified 15 commonly increased intact glycopeptides.Among them,six increased glycopeptides contain 1-2 sialic acid or core-fucosylation.And three sialylated glycopeptides were uniquely increased in multifocal tumors.In three HCC cell lines,four intact glycopeptides modified with core-fucosylation were elevated more than 2-fold at the I and/or M stages in both cell lines after quantitative analysis.Notably,two of them were identified from FOLR1.We further mapped N-linked glycans on each glycosite of FOLR1 based on our glycoproteomics data,and found that the majority of core-fucosylated intact glycopeptides were up-regulated during EMT in three cell lines.Finally,we demonstrated that FUT8 was a driver for HGF/TGF-?1-induced EMT using molecular approaches.The overall core-fucosylation and the progress of EMT were increased in FUT8-overexpresed cell lines.In addition,we also confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates,and enhanced uptake of folates further could promote the progress of EMT in HCC cells.Conclusion:Through the quantitative analysis of glycopeptides,we not only identified many commonly altered site-specific N-glycans from HCC tumors regardless of clinical stage,but also discovered site-specific glycopeptides that play an important role in the development of hepatocellular carcinoma.In addition,we confirmed a proposed novel pathway for HGF/TGF-?1-induced EMT of HCC cells: HGF/ TGF-?1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins,especially FOLR1;core-fucosylation on FOLR1 especially at the glycosite Asn-201 can then enhance the folate uptake capacity of HCC cells to promote the progression of EMT. |
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