Study On The Effects Of Caveolin-1on ST6Gal-I Expression And Adhesion Ability Of Hepatocarcinoma Cell

Primary liver cancer is one of the most common and malignant tumors. Distantorgan metastasis is the main reason for the high mortality of patients with liver cancer.The changes in the adhesion of tumor cells with the microenvironment are the initiativeand key factor for the tumor metastasis, which lead to the increased tumor cellmigration and invasion and further promote the metastasis of tumor cells to distantorgans. The adhesion of tumor cell via surface receptors such as integrins with theextracellular matrix components such as fibronectin (FN), laminin (LN) and collagen(COL) can induce the proteases secretion of tumor cells and stromal cells and furtherpromote the tumor metastasis through the lymphatic and blood stream. Thus, it iscrucial to study the adhesion mechanisms of liver cancer at molecular levels and theexpression regulation pattern of adhesion-associated factor and find valid earlydiagnosis index and treatment measures from these studies, which will make it possibleto provide new ideas and methods for the treatment of liver cancer.Protein glycosylation is a post-translational modification pattern in eukaryotes,and play an important role in the evolution of malignant tumor. Glycosylationmodification is catalyzed by the action of glycosyltransferases. Sialylation mostlyincludes α2,3-, α2,6-, α2,8-linkage form. β-galactoside:α2,6-sialyltransferase1(ST6Gal-I) is the key enzyme to synthesize the α2,6-linked sialic acid structure.ST6Gal-I catalyzes the sialic acid attached to outermost Gal of Galβ1-4GlcNAcdisaccharide units of N-glycan with a α2,6-glycosidic bond. The characteristicsincluding sialic acid taking the negative charge and its outermost position in glycanchains determine that the change in α2,6-sialylation on tumor cell surface is involved inthe regulation of tumor malignant behaviors. There is accumulating evidence thatST6Gal-I and its catalytic product α2,6-linked sialic acid were up-regulated in thecancer tissue including colon, breast, gastric, and positively regulated anti-apoptosis, angiogenesis and metastasis ability. It was reported that ST6Gal-I and α2,6-linked sialicacid expression levels in human hepatocellular carcinogenesis (HCC) tissue weresignificantly up-regulated compared with in cirrhosis and normal liver tissue, however,the regulatory mechanisms and roles involved in HCC progression remain unclear.Caveolin-1(Cav-1) is the main structural and functional components of caveolaeand involved in the regulation of cholesterol transport, endocytosis, signal transductionand tumor malignant behaviors. Studies have shown that Cav-1positively regulatesmalignant transformation, anti-apoptosis and metastasis ability of hepatocellularcarcinogenesis and plays oncogene-like role in HCC. Our previous studies showed thatCav-1up-regulated the N-glycan levels of matrix metalloproteinase inducer CD147andinduced the secretion of matrix metalloproteinases, and further promote the migrationand invasion of mouse hepatocarcinoma cell. Study indicated that Cav-1mediated thesubcompartmental localization of glycosyltransferase N-acetylglucosaminyltransferaseIII (GnT III) in human hepatocarcinoma cell Huh6. In addition, Cav-1is involved in theactivation of Wnt/beta-catenin signaling pathway, and the activated Wnt/beta-cateninsignaling can start the expression of glycosyltransferase gene DPAGT1. Thus, Cav-1may play an important role in the regulation of glycosylation.In the present study, mouse hepatocarcinoma cell with different lymphaticmetastasis ability including Hepa1-6cells (with no lymphatic metastasis potential), Hca-Fcells (with high lymphatic metastasis potential) and H22cells (with high lymphaticmetastatic potential) are as the research object, and the molecular cloning, genetransfection and RNA interference technologies et al. are used to clarify the regulationeffect of Cav-1on ST6Gal-I expression, and analyze the effect of Cav-1and ST6Gal-Ion the adhesion of cell with the extracellular matrix and lymph node, and exploremolecular mechanisms of ST6Gal-I-mediated cell adhesion to the extracellular matrixand lymph node. The study results are as follows:1. The effect of Cav-1on the expression of ST6Gal-I in mousehepatocarcinoma cellTo investigate a possible relationship between Cav-1and sialyltransferase, theCav-1and three sialyltransferases (ST6Gal-I, ST3Gal-I and ST6Gal-II) mRNAexpressions were analyzed using real-time PCR. Real-time PCR result showed that theST6Gal-I mRNA level was consistent with Cav-1being higher in Hca-F cells than inHepa1-6cells, and no significant differences in ST3Gal-I and ST6Gal-II mRNAexpression were observed between two cells. Western blot result showed that Cav-1 protein was detectable in Hca-F cells but absent in Hepa1-6cells, and ST6Gal-I proteinexpression was higher in Hca-F cells than in Hepa1-6cells. The ST6Gal-I catalyzes theformation of α2,6-linked sialic acid structure, which could be recognized by SNA lectin.SNA lectin-blot analysis revealed that Hca-F cells had higher levels of α2,6-linked sialicacid than that of Hepa1-6cells.To explore the effect of Cav-1on ST6Gal-I expression, a Hepa1-6cell line stablyexpressing Cav-1(Hepa1-6/Cav-1, Cav-1) was established. Real-time PCR and Westernblot results showed that transfection of Hepa1-6cells with Cav-1cDNA resulted in agreatly increased expression of ST6Gal-I compared with empty vector transfectedHepa1-6cells (Mock), and the transfection of ST6Gal-I-specific siRNAs intoHepa1-6/Cav-1cells significantly inhibited the expression of ST6Gal-I. SNA lectin-blotresult indicated that Cav-1overexpression up-regulated the levels of α2,6-linked sialicacid in Hepa1-6cells, which were reduced by the transfection of the ST6Gal-I-specificsiRNAs into Hepa1-6/Cav-1cells.To further verify the effect of Cav-1on ST6Gal-I expression, we developed threeshRNA interference vectors (shCav-1-1, shCav-1-2and shCav-1-3) to silence Cav-1expression in Hca-F cells, respectively. Real-time PCR and Western blot results showedthat Cav-1and ST6Gal-I expression were suppressed in different Cav-1-shRNAtransfected cells compared with the negative control (shNC) transfected cells, andshCav-1-1was more efficient in down-regulation the expression of Cav-1and ST6Gal-Ithan shCav-1-2or shCav-1-3. The reduced levels of ST6Gal-I in shCav-1-1transfectedHca-F cells were recovered by the reintroduction of wild-type Cav-1or ST6Gal-I.α2,6-linked sialic acid levels were reduced in Cav-1shRNA transfectants comparedwith control transfectants, and the re-expression of wild-type Cav-1or ST6Gal-I inCav-1knockdown cells significantly restored the α2,6-linked sialic acid levels revealedby SNA lectin-blot.To determine which domain of Cav-1regulates ST6Gal-I expression, the Cav-1palmitoylation mutant or Cav-1scaffolding domain (CSD) mutant was transfected intoCav-1knockdown Hca-F cells. Real-time PCR, Western blot and SNA lectin-blotresults showed that the ST6Gal-I mRNA, protein and α2,6-linked sialic acid levels weresignificantly increased in palmitoylation mutant but not CSD mutant transfected Cav-1knockdown cells compared with un-transfected knockdown cells.To investigate whether Cav-1influences ST6Gal-I expression at transcriptionlevel, we obtained ST6Gal-I promoter region using the UCSC Genome Browser (http://genome.ucsc.edu) and constructed the recombinant luciferase vectorpGL3-basic/ST6Gal-I. Real-time PCR and Western blot results showed that thetransfection of three RNAi vectors shCav-1-1, shCav-1-2and shCav-1-3into H22cellsobviously inhibited the expression of ST6Gal-I, respectively, and shCav-1-1have themost obvious effectiveness to down-regulate the expression of ST6Gal-I, which wererestored by the introduction of wild-type Cav-1or ST6Gal-I. Promoter activity assayresults revealed that shCav-1-1transfectant exhibited a weaker luciferase activity drivenby ST6Gal-I promoter compared with shCav-1-2or shCav-1-3transfectant, and thetransfection of Cav-1into Cav-1-silenced cells resulted in a notable restoration ofST6Gal-I promoter controlled luciferase activity.2. The effects of Cav-1and ST6Gal-I on the adhesion of mousehepatocarcinoma cell to extracellular matrix or lymph nodeIn vitro cell adhesion assay results showed that Hca-F cells exhibited significantlyhigher adhesion to ECM components including FN, COL or LN in comparison toHepa1-6cells.In vitro cell adhesion assay and Western blot results showed that the transfectionof Hepa1-6cells with Cav-1cDNA resulted in a greatly enhanced adhesion of cells toFN and increased FN-mediated FAK phosphorylation compared with Mock transfectedHepa1-6cell, which were inhibited by the transfection of ST6Gal-I-siRNAs intoHepa1-6/Cav-1cells. Conversely, cell adhesion assay showed that the reduced adhesionof cells to FN, resulted by Cav-1silencing, was rescued by the transfection of wild-typeCav-1or ST6Gal-I into Cav-1knockdown Hca-F cells. Western blot results revealedthat the phosphorylation levels of FAK were remarkably recovered in Cav-1-orST6Gal-I-rescued cells compared with in Cav-1knockdown cells.In vitro and vivo lymph node adhesion results showed that the transfection ofST6Gal-I-specific siRNAs suppressed the adhesion of Hca-F cells to lymph node.3. The molecule mechanisms of ST6Gal-I regulating the adhesion of mousehepatocarcinoma cell to extracellular matrix or lymph nodeSNA lectin staining and flow cytometry results showed that the levels ofα2,6-linked sialic acid on H22cell surface were notably decreased in shCav-1-1transfectant compared with negative control transfectant, and Cav-1-orST6Gal-I-rescued cells showed a significant increase in the levels of cell surfaceα2,6-linked sialic acid compared with non-rescued cells. In vitro cell adhesion assayresults revealed that the transfection of shCav-1-1into H22cells greatly reduced the cell adhesion to FN compared with negative control transfected cells, and no obviousdifference was observed in the adhesion of both cells to COL or LN. The re-expressionof wild-type ST6Gal-I in Cav-1knockdown H22cells significantly restored the reducedadhesion of Cav-1-silenced cells. After the treatment with FN, phosphorylated FAK andpaxillin were notably down-regulated in shCav-1-1transfectant compared with controltransfectant and were remarkably recovered in Cav-1-or ST6Gal-I-rescued Cav-1knockdown cells as revealed by Western blot.The α5-subunit was immunoprecipitated from the total membrane protein ofCav-1-rescued, ST6Gal-I-rescued, shCav-1-1or control transfectant, and then probedwith SNA lectin or anti-α5subunit antibody. Immunoprecipitation results showed thatthe transfection of shCav-1-1into H22cells resulted in a decrease in the α2,6-linkedsialic acid on α5-subunit, which could be restored by the re-expression of Cav-1orST6Gal-I in shCav-1-1transfectant. Cav-1and ST6Gal-I had no significant effect onthe expression of cell surface α5-or β1-subunit as revealed by SNA lectin-blot and flowcytometry assay. Adhesion inhibition assay using function-blocking antibody resultsshowed that the adhesion of non-rescued Cav-1knockdown cells was more prone to beblocked as compared to that of ST6Gal-I-rescued cells in the presence of anti-α5oranti-β1subunit antibody.In vitro cell adhesion assay results revealed that the transfection of ST6Gal-IsiRNAs significantly inhibited the adhesion of Hca-F cells to CD22compared with thatof control siRNA. The adhesion of Hca-F cells to lymph node was inhibited by thedown-regulation of ST6Gal-I, and ST6Gal-I siRNAs transfected cells were more proneto be blocked as compared to control siRNA transfected cells in the presence ofanti-CD22antibody as revealed by lymph node adhesion inhibition assay usingfunction-blocking antibody.In conclusion, we demonstrates for the first time that caveolin-1can up-regulateST6Gal-I expression at transcription level, and Cav-1scaffolding domain is involved inthis regulation. Cav-1-induced up-regulation of ST6Gal-I contributes to promotingmouse hepatocarcinoma cell adhesion to fibronectin and lymph node. Cell surfaceα2,6-sialylation is required for cell adhesion to fibronectin, and α2,6-sialylatedα5-subunit mediates integrin α5β1-dependent cell adhesion. α2,6-linked sialic acid viarecognizing CD22in lymph node facilitates the adhesion of mouse hepatocarcinomacell to lymph node. This study provides new insights into the biological functions ofCav-1and the significance of sialylation modification in HCC metastasis.