Caveolin-1调控肝癌细胞ST6Gal-I表达及对细胞黏附的影响研究
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摘要
原发性肝癌是我国最常见且恶性程度最高的肿瘤之一，远隔器官转移是肝癌患者致死率高的主要原因。肿瘤细胞与肿瘤微环境黏附能力的改变是导致肿瘤细胞迁移和侵袭能力增加进而促进肿瘤细胞向远隔器官转移的始动和关键因素。肿瘤细胞首先通过膜表面受体如整合素（integrin）与细胞外基质成分如纤连蛋白（Fibronectin，FN）、层粘连蛋白（Laminin，LN）、胶原蛋白（Collagen，COL）的黏附，诱导肿瘤细胞及基质细胞释放蛋白酶降解基底膜和基质，促使肿瘤细胞经血道和淋巴道转移。因此，从分子水平研究肝癌黏附机制，探寻黏附相关因子的表达调控规律，并针对此寻找有效的早期诊断指标及治疗措施将可能为肝癌的治疗提供新的思路和方法。
蛋白质糖基化是真核生物体内重要的翻译后修饰方式，其在恶性肿瘤的演变过程发挥重要作用。唾液酸化（sialylation）作为一种糖基化形式，主要包括α2,3-，α2,6-，α2,8-三种连接方式。唾液酰基转移酶β-galactoside:α2-6-sialyltransferase1（ST6Gal-I）是催化唾液酸以α2,6-糖苷键与N-聚糖最外侧Galβ1-4GlcNAc二糖单位中Gal连接的关键酶，形成α2,6连接唾液酸（α2,6-linked sialic acid）结构。由于唾液酸本身带负电荷的特性及所处聚糖链最外侧的位置特点，决定肿瘤细胞表面唾液酸化改变参与调控肿瘤恶性行为。有证据表明，ST6Gal-I及其催化产物α2,6连接唾液酸在包括结肠癌、乳腺癌、胃癌等癌组织中表达上调，而且正向调控癌细胞的抗凋亡，促血管生成以及转移能力。研究发现，ST6Gal-I及α2,6连接唾液酸水平在肝癌组织中较慢性肝疾病及正常肝组织中明显增加，然而其上调的调控机制及在肝癌进展中发挥的作用还不明确。
窖蛋白-1（caveolin-1，Cav-1）是胞膜窖的主要结构和功能成分，参与调控胆固醇转运、细胞内吞、信号转导以及肿瘤的恶性行为。已有研究表明，Cav-1促进肝癌细胞的恶性转化、抗凋亡和转移等能力，在肝癌中发挥癌基因样作用。我们实验室前期研究发现，Cav-1通过上调基质金属蛋白酶诱导剂CD147的N-聚糖水平，从而诱导基质金属蛋白酶的分泌，进一步促进小鼠肝癌细胞的迁移和侵袭。有证据显示，Cav-1能够介导糖基转移酶N-acetylglucosaminyltransferase III（GnTIII）在人肝癌细胞Huh6中的高尔基体定位。此外，Cav-1参与活化Wnt/beta-catenin信号通路，而活化的Wnt/beta-catenin信号能够启动糖基转移酶基因DPAGT1的表达。因此，Cav-1有可能对糖基化修饰发挥重要的调控作用。
本文以不同淋巴道转移能力的小鼠肝癌Hepa1-6细胞（无淋巴道转移潜能）、Hca-F细胞（高淋巴道转移潜能）和H22细胞（高淋巴道转移潜能）等为研究对象，应用分子克隆、基因转染、RNA干扰等技术，1）阐明Cav-1对ST6Gal-I表达的调控作用；2）分析Cav-1及ST6Gal-I对细胞与细胞外基质及淋巴结黏附的影响；3）探讨ST6Gal-I介导细胞与细胞外基质及淋巴结黏附的分子机制。本研究实验结果如下：
1. Cav-1对小鼠肝癌细胞ST6Gal-I表达的调控
（1）分析Cav-1及三种唾液酰基转移酶在小鼠肝癌细胞Hca-F和Hepa1-6中的表达
1）Real-time PCR结果显示：Hca-F细胞高表达Cav-1及ST6Gal-I，Hepa1-6细胞未检测到Cav-1表达并低表达ST6Gal-I，两种细胞的ST3Gal-I及ST6Gal-II的表达丰度没有明显差别；2）Western blot检测到的两种细胞内Cav-1及ST6Gal-I的表达趋势与Real-time PCR结果基本一致；3）SNA凝集素能够特异性识别ST6Gal-I的催化产物，即α2,6连接唾液酸结构，SNA凝集素印记结果显示：Hca-F细胞的α2,6连接唾液酸水平明显高于Hepa1-6细胞。
（2）过表达Cav-1上调Hepa1-6细胞的ST6Gal-I表达
1）Real-time PCR和Western blot结果显示：稳定表达Cav-1的Hepa1-6细胞（Hepa1-6/Cav-1，Cav-1）与转染空载体的Hepa1-6细胞（Mock）相比，ST6Gal-I的表达明显增加，ST6Gal-I特异性siRNAs（ST6Gal-I-siRNAs）能够显著下调Hepa1-6/Cav-1细胞的ST6Gal-I表达；2）SNA凝集素印记结果显示：Cav-1的稳定表达上调了Hepa1-6细胞的α2,6连接唾液酸水平，其能够被ST6Gal-I-siRNAs所抑制。
（3）Cav-1沉默下调了Hca-F细胞的ST6Gal-I表达，其能够被野生型Cav-1及ST6Gal-I的重新引入所恢复
1）将Cav-1特异性短发夹RNA（shRNA）干扰载体（shCav-1-1、shCav-1-2和shCav-1-3）分别转染Hca-F细胞，Real-time PCR及Western blot结果显示：与阴性对照（shNC）相比，三种干扰载体中，以shCav-1-1下调Cav-1的效果最明显，同时对ST6Gal表达的抑制作用也最显著，shCav-1-1对ST6Gal-I的抑制能够被野生型Cav-1及ST6Gal-I的重新引入所恢复；2）SNA凝集素印记结果显示：shCav-1-1的转染明显下调了Hca-F细胞的α2,6连接唾液酸水平，其能够被野生型Cav-1及ST6Gal-I的转染所逆转。
（4）Cav-1通过其绞手架结构域（CSD）上调ST6Gal-I表达
利用Cav-1绞手架结构域突变体（△C SD）及Cav-1第133、143、156位棕榈酰化突变体（△C133,143,156A）分别转染Cav-1沉默的Hca-F细胞，Real-timePCR、Western blot及SNA凝集素印记结果显示：△C133,143,156A能够营救ST6Gal-I表达及α2,6连接唾液酸水平，而△CSD不能发挥上述作用。
（5）Cav-1通过诱导ST6Gal-I启动子活性促进H22细胞的ST6Gal-I表达
1）将shCav-1-1、shCav-1-2和shCav-1-3三种干扰载体分别转染H22细胞，Real-time PCR、Western blot结果显示，三种干扰载体中以shCav-1-1对Cav-1的沉默效果最明显，同时对ST6Gal-I表达的抑制也最显著，shCav-1-1对ST6Gal-I的抑制作用能够被野生型Cav-1及ST6Gal-I的重新引入所恢复；2）利用UCSCGenome Browser (http://genome.ucsc.edu)获得ST6Gal-I的启动子区，并构建重组荧光素酶报告载体pGL3-basic/ST6Gal-I。荧光素酶活性检测结果显示：三种Cav-1干扰载体分别转染H22细胞后，减弱了ST6Gal-I启动子控制的荧光素酶活性，以shCav-1-1的抑制效果最显著，其能够被野生型Cav-1的营救所逆转。
2. Cav-1、ST6Gal-I对小鼠肝癌细胞与细胞外基质及淋巴结黏附的影响
（1）检测小鼠肝癌Hca-F和Hepa1-6细胞与FN、COL及LN的黏附
体外细胞黏附结果显示：Hca-F细胞与细胞外基质主要成分FN、COL、LN的黏附能力明显高于Hepa1-6细胞。
（2）Cav-1过表达上调Hepa1-6细胞与FN的黏附及FAK信号，其能够被ST6Gal-I的下调所抑制
体外细胞黏附及Western blot结果显示：与Mock转染相比，Cav-1的转染显著上调了Hepa1-6细胞与FN的黏附及黏附信号分子FAK的磷酸化水平，其能够被ST6Gal-I-siRNAs所抑制，而且Cav-1过表达没有影响Hepa1-6细胞与COL或LN的黏附。
（3）下调Cav-1表达抑制Hca-F细胞与FN的黏附及FAK信号，其能够被野生型Cav-1或ST6Gal-I的重新引入所恢复
体外细胞黏附及Western blot结果显示：与shNC相比，Cav-1沉默减少了Hca-F细胞与FN的黏附及磷酸化FAK水平，其能够被野生型Cav-1及ST6Gal-I的转染所营救，Cav-1表达下调没有影响Hca-F细胞与COL或LN的黏附。
（4）下调ST6Gal-I表达抑制Hca-F细胞与淋巴结的黏附
体外及体内淋巴结黏附结果显示：ST6Gal-I-siRNAs的转染显著减少了Hca-F细胞与淋巴结的黏附。
3. ST6Gal-I调控小鼠肝癌细胞与细胞外基质及淋巴结黏附的分子机制
（1）Cav-1上调H22细胞表面α2,6连接唾液酸水平，细胞表面α2,6唾液酸化促进了H22细胞与FN的黏附及FAK信号的活化
1）SNA凝集素印记和流式细胞技术结果显示：Cav-1特异性shRNA的转染减少了H22细胞表面α2,6连接唾液酸水平，其能够被野生型Cav-1及ST6Gal-I的转染所恢复；2）体外细胞黏附结果显示：Cav-1沉默抑制了H22细胞与FN的黏附，其能够被野生型ST6Gal-I的引入所恢复，Cav-1沉默没有影响H22细胞与COL或LN的黏附；3）Western blot结果显示：Cav-1下调抑制了H22细胞的FAK和Paxillin磷酸化，野生型Cav-1及ST6Gal-I的转染逆转了上述变化。
（2）α5-integrin的α2,6唾液酸化介导了H22细胞与FN的黏附
1）免疫沉淀结果显示：Cav-1沉默下调了H22细胞表面α5-integrin的α2,6唾液酸化水平，其能够被野生型Cav-1及ST6Gal-I的重新引入所恢复；2）Westernblot和流式细胞技术结果显示：Cav-1和ST6Gal-I没有影响H22细胞表面integrinα5β1的表达；3）体外细胞黏附的功能性抗体阻断结果显示：与ST6Gal-I营救的Cav-1沉默细胞相比，Cav-1沉默细胞与FN的黏附更容易被抗-α5或抗-β1整合素抗体所阻断。
（3）ɑ2,6连接唾液酸与CD22的识别介导Hca-F细胞与淋巴结黏附
1）体外细胞黏附结果显示：与Control-siRNA的转染相比，ST6Gal-I-siRNAs明显抑制了Hca-F细胞与CD22的黏附；2）体外淋巴结黏附的功能性抗体阻断结果显示：在不同浓度的抗-CD22抗体作用下，与对应的Control-siRNA转染细胞相比，ST6Gal-I-siRNAs转染的Hca-F细胞与淋巴结的黏附更容易被抗体阻断。
结论
1. Cav-1在转录水平促进小鼠肝癌细胞的ST6Gal-I表达，Cav-1的CSD区参与该调控过程；
2. Cav-1诱导的ST6Gal-I表达介导了小鼠肝癌细胞与FN及淋巴结的黏附；
3.小鼠肝癌细胞表面α5-integrin的α2,6连接唾液酸化介导了integrin α5β1依赖的细胞黏附及FAK信号，α2,6连接唾液酸与CD22的识别参与小鼠肝癌细胞与淋巴结的黏附。
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 cell
To 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 node
In 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 node
SNA 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.
蛋白质糖基化是真核生物体内重要的翻译后修饰方式，其在恶性肿瘤的演变过程发挥重要作用。唾液酸化（sialylation）作为一种糖基化形式，主要包括α2,3-，α2,6-，α2,8-三种连接方式。唾液酰基转移酶β-galactoside:α2-6-sialyltransferase1（ST6Gal-I）是催化唾液酸以α2,6-糖苷键与N-聚糖最外侧Galβ1-4GlcNAc二糖单位中Gal连接的关键酶，形成α2,6连接唾液酸（α2,6-linked sialic acid）结构。由于唾液酸本身带负电荷的特性及所处聚糖链最外侧的位置特点，决定肿瘤细胞表面唾液酸化改变参与调控肿瘤恶性行为。有证据表明，ST6Gal-I及其催化产物α2,6连接唾液酸在包括结肠癌、乳腺癌、胃癌等癌组织中表达上调，而且正向调控癌细胞的抗凋亡，促血管生成以及转移能力。研究发现，ST6Gal-I及α2,6连接唾液酸水平在肝癌组织中较慢性肝疾病及正常肝组织中明显增加，然而其上调的调控机制及在肝癌进展中发挥的作用还不明确。
窖蛋白-1（caveolin-1，Cav-1）是胞膜窖的主要结构和功能成分，参与调控胆固醇转运、细胞内吞、信号转导以及肿瘤的恶性行为。已有研究表明，Cav-1促进肝癌细胞的恶性转化、抗凋亡和转移等能力，在肝癌中发挥癌基因样作用。我们实验室前期研究发现，Cav-1通过上调基质金属蛋白酶诱导剂CD147的N-聚糖水平，从而诱导基质金属蛋白酶的分泌，进一步促进小鼠肝癌细胞的迁移和侵袭。有证据显示，Cav-1能够介导糖基转移酶N-acetylglucosaminyltransferase III（GnTIII）在人肝癌细胞Huh6中的高尔基体定位。此外，Cav-1参与活化Wnt/beta-catenin信号通路，而活化的Wnt/beta-catenin信号能够启动糖基转移酶基因DPAGT1的表达。因此，Cav-1有可能对糖基化修饰发挥重要的调控作用。
本文以不同淋巴道转移能力的小鼠肝癌Hepa1-6细胞（无淋巴道转移潜能）、Hca-F细胞（高淋巴道转移潜能）和H22细胞（高淋巴道转移潜能）等为研究对象，应用分子克隆、基因转染、RNA干扰等技术，1）阐明Cav-1对ST6Gal-I表达的调控作用；2）分析Cav-1及ST6Gal-I对细胞与细胞外基质及淋巴结黏附的影响；3）探讨ST6Gal-I介导细胞与细胞外基质及淋巴结黏附的分子机制。本研究实验结果如下：
1. Cav-1对小鼠肝癌细胞ST6Gal-I表达的调控
（1）分析Cav-1及三种唾液酰基转移酶在小鼠肝癌细胞Hca-F和Hepa1-6中的表达
1）Real-time PCR结果显示：Hca-F细胞高表达Cav-1及ST6Gal-I，Hepa1-6细胞未检测到Cav-1表达并低表达ST6Gal-I，两种细胞的ST3Gal-I及ST6Gal-II的表达丰度没有明显差别；2）Western blot检测到的两种细胞内Cav-1及ST6Gal-I的表达趋势与Real-time PCR结果基本一致；3）SNA凝集素能够特异性识别ST6Gal-I的催化产物，即α2,6连接唾液酸结构，SNA凝集素印记结果显示：Hca-F细胞的α2,6连接唾液酸水平明显高于Hepa1-6细胞。
（2）过表达Cav-1上调Hepa1-6细胞的ST6Gal-I表达
1）Real-time PCR和Western blot结果显示：稳定表达Cav-1的Hepa1-6细胞（Hepa1-6/Cav-1，Cav-1）与转染空载体的Hepa1-6细胞（Mock）相比，ST6Gal-I的表达明显增加，ST6Gal-I特异性siRNAs（ST6Gal-I-siRNAs）能够显著下调Hepa1-6/Cav-1细胞的ST6Gal-I表达；2）SNA凝集素印记结果显示：Cav-1的稳定表达上调了Hepa1-6细胞的α2,6连接唾液酸水平，其能够被ST6Gal-I-siRNAs所抑制。
（3）Cav-1沉默下调了Hca-F细胞的ST6Gal-I表达，其能够被野生型Cav-1及ST6Gal-I的重新引入所恢复
1）将Cav-1特异性短发夹RNA（shRNA）干扰载体（shCav-1-1、shCav-1-2和shCav-1-3）分别转染Hca-F细胞，Real-time PCR及Western blot结果显示：与阴性对照（shNC）相比，三种干扰载体中，以shCav-1-1下调Cav-1的效果最明显，同时对ST6Gal表达的抑制作用也最显著，shCav-1-1对ST6Gal-I的抑制能够被野生型Cav-1及ST6Gal-I的重新引入所恢复；2）SNA凝集素印记结果显示：shCav-1-1的转染明显下调了Hca-F细胞的α2,6连接唾液酸水平，其能够被野生型Cav-1及ST6Gal-I的转染所逆转。
（4）Cav-1通过其绞手架结构域（CSD）上调ST6Gal-I表达
利用Cav-1绞手架结构域突变体（△C SD）及Cav-1第133、143、156位棕榈酰化突变体（△C133,143,156A）分别转染Cav-1沉默的Hca-F细胞，Real-timePCR、Western blot及SNA凝集素印记结果显示：△C133,143,156A能够营救ST6Gal-I表达及α2,6连接唾液酸水平，而△CSD不能发挥上述作用。
（5）Cav-1通过诱导ST6Gal-I启动子活性促进H22细胞的ST6Gal-I表达
1）将shCav-1-1、shCav-1-2和shCav-1-3三种干扰载体分别转染H22细胞，Real-time PCR、Western blot结果显示，三种干扰载体中以shCav-1-1对Cav-1的沉默效果最明显，同时对ST6Gal-I表达的抑制也最显著，shCav-1-1对ST6Gal-I的抑制作用能够被野生型Cav-1及ST6Gal-I的重新引入所恢复；2）利用UCSCGenome Browser (http://genome.ucsc.edu)获得ST6Gal-I的启动子区，并构建重组荧光素酶报告载体pGL3-basic/ST6Gal-I。荧光素酶活性检测结果显示：三种Cav-1干扰载体分别转染H22细胞后，减弱了ST6Gal-I启动子控制的荧光素酶活性，以shCav-1-1的抑制效果最显著，其能够被野生型Cav-1的营救所逆转。
2. Cav-1、ST6Gal-I对小鼠肝癌细胞与细胞外基质及淋巴结黏附的影响
（1）检测小鼠肝癌Hca-F和Hepa1-6细胞与FN、COL及LN的黏附
体外细胞黏附结果显示：Hca-F细胞与细胞外基质主要成分FN、COL、LN的黏附能力明显高于Hepa1-6细胞。
（2）Cav-1过表达上调Hepa1-6细胞与FN的黏附及FAK信号，其能够被ST6Gal-I的下调所抑制
体外细胞黏附及Western blot结果显示：与Mock转染相比，Cav-1的转染显著上调了Hepa1-6细胞与FN的黏附及黏附信号分子FAK的磷酸化水平，其能够被ST6Gal-I-siRNAs所抑制，而且Cav-1过表达没有影响Hepa1-6细胞与COL或LN的黏附。
（3）下调Cav-1表达抑制Hca-F细胞与FN的黏附及FAK信号，其能够被野生型Cav-1或ST6Gal-I的重新引入所恢复
体外细胞黏附及Western blot结果显示：与shNC相比，Cav-1沉默减少了Hca-F细胞与FN的黏附及磷酸化FAK水平，其能够被野生型Cav-1及ST6Gal-I的转染所营救，Cav-1表达下调没有影响Hca-F细胞与COL或LN的黏附。
（4）下调ST6Gal-I表达抑制Hca-F细胞与淋巴结的黏附
体外及体内淋巴结黏附结果显示：ST6Gal-I-siRNAs的转染显著减少了Hca-F细胞与淋巴结的黏附。
3. ST6Gal-I调控小鼠肝癌细胞与细胞外基质及淋巴结黏附的分子机制
（1）Cav-1上调H22细胞表面α2,6连接唾液酸水平，细胞表面α2,6唾液酸化促进了H22细胞与FN的黏附及FAK信号的活化
1）SNA凝集素印记和流式细胞技术结果显示：Cav-1特异性shRNA的转染减少了H22细胞表面α2,6连接唾液酸水平，其能够被野生型Cav-1及ST6Gal-I的转染所恢复；2）体外细胞黏附结果显示：Cav-1沉默抑制了H22细胞与FN的黏附，其能够被野生型ST6Gal-I的引入所恢复，Cav-1沉默没有影响H22细胞与COL或LN的黏附；3）Western blot结果显示：Cav-1下调抑制了H22细胞的FAK和Paxillin磷酸化，野生型Cav-1及ST6Gal-I的转染逆转了上述变化。
（2）α5-integrin的α2,6唾液酸化介导了H22细胞与FN的黏附
1）免疫沉淀结果显示：Cav-1沉默下调了H22细胞表面α5-integrin的α2,6唾液酸化水平，其能够被野生型Cav-1及ST6Gal-I的重新引入所恢复；2）Westernblot和流式细胞技术结果显示：Cav-1和ST6Gal-I没有影响H22细胞表面integrinα5β1的表达；3）体外细胞黏附的功能性抗体阻断结果显示：与ST6Gal-I营救的Cav-1沉默细胞相比，Cav-1沉默细胞与FN的黏附更容易被抗-α5或抗-β1整合素抗体所阻断。
（3）ɑ2,6连接唾液酸与CD22的识别介导Hca-F细胞与淋巴结黏附
1）体外细胞黏附结果显示：与Control-siRNA的转染相比，ST6Gal-I-siRNAs明显抑制了Hca-F细胞与CD22的黏附；2）体外淋巴结黏附的功能性抗体阻断结果显示：在不同浓度的抗-CD22抗体作用下，与对应的Control-siRNA转染细胞相比，ST6Gal-I-siRNAs转染的Hca-F细胞与淋巴结的黏附更容易被抗体阻断。
结论
1. Cav-1在转录水平促进小鼠肝癌细胞的ST6Gal-I表达，Cav-1的CSD区参与该调控过程；
2. Cav-1诱导的ST6Gal-I表达介导了小鼠肝癌细胞与FN及淋巴结的黏附；
3.小鼠肝癌细胞表面α5-integrin的α2,6连接唾液酸化介导了integrin α5β1依赖的细胞黏附及FAK信号，α2,6连接唾液酸与CD22的识别参与小鼠肝癌细胞与淋巴结的黏附。
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 cell
To 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 node
In 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 node
SNA 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.
引文
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