| Glycosphingolipids(GSLs)are ubiquitous cell components distributing on cell membrane and act as crucial roles in many biological processes such as cell adhesion,cell growth,apoptosis,proliferation,angiogenesis,inflammatory response,as well as intracellular homeostasis.GSLs are composed of a glycan structure extending into the extracellular matrix and a lipid moiety anchoring the outer leaflet of cell membranes.This combination makes GSL an amphiphilic molecule.GSLs are highly related to some diseases such as cancer and the identification of unknown GSL-glycan structures is still a major challenge.In 2018,liver cancer ranks 6th in the most diagnosed cancer and it has the 4th highest mortality globally and even higher in China.90% of liver cancer incidence in China is hepatocellular carcinoma(HCC).It is reported that GSL showed abnormal expression in various cancer tissues and cell lines while the exact mechanism of how GSL-glycan synthesis change during the development of cancer is still unclear.In this study,one human normal liver cell line(HL-7702)and three human hepatocarcinoma cell lines(MHCC97L,MHCC97 H,and HCCLM3)were used to decipher the cell phenotype in terms of the glycopattern of GSL-glycan and to characterize detailed GSL-glycan structures in HCC cell lines.Furthermore,glycogenes were studied in HCC cell lines and TCGA analysis was carried out to determine the exact mechanism of GSL-glycan synthesis during HCC development and the correlation between GSL-glycan synthesis and HCC cell lines with different metastasis potential was established.In the first chapter of this paper,the general information of GSL,the development of GSL research technique,and some discoveries underlying GSL and GSL-related diseases were discussed.Furthermore,the progress and prospect of glycomics in GSL research,and the preliminary research related to this topic in our laboratory were reviewed.Chapter 2 of this study was to utilize the method of the analysis of GSL-glycan by lectin microarray to conduct the glycopatterns of GSL from three HCC cell lines(MHCC97L,MHCC97 H,and HCCLM3)and normal control(HL-7702).This glycomics research was started by analyzing the glycopatterns of a standard ganglioside GM1 a via lectin microarray.Negative test of GM1 a showed that using lectin microarrays to analyze GSL-glycan was a methodology which was lower cost,high-throughput,and easy operation.The analysis of GSL-glycans from HCC cell lines showed that there were 27lectins(e.g.,WFA,MAL-II,and LTL)to give significantly different signals compared to normal human liver cell line.To be specific,13 lectins(e.g.,DSA,DBA,and HHL)exhibited increased NFIs in HMCC97 L compared with HL-7702(all fold changes ≥1.54,P< 0.05);on the other hand,6 lectins(e.g.,AAL,WFA,and LCA)showed significantly decreased NFIs in HMCC97 L compared with HL-7702(all fold changes ≤0.63,P < 0.001).Similarly,there were 13 lectins(e.g.,PSA,PTL-I,and MAL-II)that showed significantly increased NFIs(all fold changes ≥1.55,P < 0.05),and 5 lectins(e.g.,ECA,LCA,and AAL)that showed significantly decreased NFIs(all fold changes ≤0.60,P <0.001)in HMCC97 H compared with HL-7702.There were 11 lectins(e.g.,SJA,SBA,and PNA)that showed significantly increased NFIs(all fold changes ≥1.51,P < 0.01),and 7lectins(e.g.,WFA,GSL-II,and NPA)that showed significantly decreased NFIs(all fold changes ≤0.60,P < 0.01)in HCCLM3 compared with HL-7702.Chapter 3 of this study was to utilize the NaClO oxidative release of GSL-glycans for the first time in a lab-scale(~108cells),and the analysis and characterize of GSL-glycans was carried out by Matrix-Assisted Laser Desorption/Ionization Time-ofFlight Mass Spectrometry and a tandem MS/MS.The results showed that A total of11,11,11,and 15 GSL-glycan signals were identified and annotated with proposed structures from HL-7702,MHCC97 L,MHCC97H,and HCCLM3 cell lines according to the results of lectin microarrays,respectively.Of these,there were 6 glycan signals(m/z 728.211 [Galβ1-3Galα1-4Galβ1-4Glc],747.256 [Galβ1-3GlcNAcβ1-3Galβ1-4Glc],876.298 [GalNAcβ1-4(Neu Acα2-3)Galβ1-4Glc],893.314 [Galβ1-3(Fucα1-4)G lcNAcβ1-3Galβ1-4Glc and/or Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1038.351 [Galβ1-3GalNAcβ1-4(Neu Acα2-3)Galβ1-4Glc],and 1055.366 [Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc] that to be present in all cell lines with different intensities.9 glycan signals(m/z 980.309 [Neu Gcα2-8Neu Acα2-3Galβ1-4Glc],1096.393 [GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1112.388 [Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1134.370 [Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1201.424 [Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],1242.451 [GalNAcα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],1286.348 [Neu Acα2-3S ulf Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],1550.562 [Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],and 1712.614 [Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc])were present only in HCC cell lines,7 glycan signals(m/z 1096.393 [GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1112.388 [Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1201.424 [Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],1242.451[GalNAcα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],1286.348 [Neu Acα2-3Sulf Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],1550.562 [Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],and 1712.614 [Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc])were present only in MHCC97 H and/or HCCLM3 cell lines with high metastasis potential.There was a signal at m/z 1280.428 [Galβ1-3GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc] that was unique in HL-7702 cell line,1 glycan signal(m/z 1134.370[Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc])was unique in MHCC97 L cell line with low metastatic potential,and 5 glycan signals(m/z 1096.393 [GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1112.388 [Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc],1201.424 [Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],1242.451 [GalNAcα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc],and 1286.348 [Neu Acα2-3Sulf Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc])were unique in HCCLM3 cell line with high metastatic potential.Chapter 4 of this study was to analyze the expression profile of glycan-related genes in HCC cell lines using glycogene microarray,and the quantitative real-time PCR was used to verify the results of glycogene microarray.256 ± 24(63.68% of the total probes)probes showed valid signals in the glycogene expression profile analysis.There were 37(e.g.,CTSA,HYAL2,and AHSG)glycogenes showed up-regulations in HCC cell lines with high and low potential of metastasis compared with HL-7702,and 57(e.g.,AGA,B3GALT5,and COX7C)glycogenes showed down-regulations in HCC cell lines with high and low potential of metastasis compared with HL-7702.Several GSL-related glycogenes were screened and 4 genes were verified by q RT-PCR validation.The TCGA data set analysis was carried out to discover the exact mechanism during the development of HCC.By combining the results of Chapter 2 as well as Chapter 3,the results of the integrated analysis showed that the biosynthesis and metabolism of sphingolipids remained unchanged during the development of HCC,the biosynthesis of Siaα2-3 linked GSL glycans in the Ganlio-series GSL up-regulated along with the metastasis ability of HCC cell rise,furthermore,the biosynthesis of Fucα1-2 linked GSL-glycans and Siaα2-3 linked GSL glycans in the Globo-series GSL also up-regulated along with the rise of metastasis ability of HCC cell rise. |
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