探討B3GNT7與IGnTA醣轉移酵素基因 於大腸癌細胞轉移的角色與表現機制

Abstract

消化道系統相關癌症癌化的過程會伴隨癌細胞表面醣結構的改變，這些特定出現於癌細胞表面的醣稱為腫瘤相關醣抗原(tumor-associated antigens)；sialyl Lewis a (sLea)與sialyl Lewis x (sLex)是最著名的消化道系統相關癌症的腫瘤相關醣抗原。過去的研究證實，sLea與sLex會促進消化道系統相關癌症的癌細胞轉移(metastasis)，且此二醣抗原的表現和術後癌症復發與病人存活率有很大的關係。sLea與sLex建構於重複的N-acetyllactosamine (Gal-GlcNAc，LacNAc)單元所組成的poly-LacNAc鏈的末端。本實驗室過去的研究發現，大腸癌病患的大腸腫瘤組織中，兩個參與poly-LacNAc鏈建構的基因，β-1,3-N-acetylglucosaminyltransferase 7 (B3GNT7)與I-branching β-1,6-N-acetylglucosaminyltransferase (IGnTA)，其表現相較於正常大腸組織降低許多。因此， poly-LacNAc鏈的生合成出現問題，是否會改變sLea與sLex的表現，進而影響大腸癌細胞轉移，是我們想要探討的問題。 本研究以大腸癌細胞株做為研究的細胞模型，探討B3GNT7與IGnTA的表現對於大腸癌細胞轉移的影響，並進一步探討造成此二基因於大腸癌細胞中表現降低的機制。我們的研究發現，大量表現B3GNT7與IGnTA的大腸癌細胞，會分別降低其細胞表面sLex與sLea的表現；裸鼠實驗中，大量表現B3GNT7與IGnTA的大腸癌細胞，其癌細胞轉移的情況相較於對照組降低許多。我們也發現大腸癌細胞的B3GNT7與IGnTA的5端調控區域有高度甲基化的現象，並且利用去甲基化藥物5-Aza-2’-deoxycytidine (5AzadC)可回復大腸癌細胞中此二基因的表現與降低B3GNT7與IGnTA的5端調控區域的甲基化程度，顯示大腸癌細胞調控此二基因表現的機制與DNA甲基化有很大的關係。進一步地，我們發現利用epigenetic藥物，包括5AzadC與組蛋白去乙醯酵素抑制劑(HDAC inhibitors)，可增加大腸癌細胞中IGnTA的表現，並降低其細胞表面sLea的表現。 我們的研究證實，大腸癌細胞中B3GNT7與IGnTA表現降低的原因與DNA甲基化有很大的關係，而此二基因表現的改變，將影響細胞表面醣抗原的表現，進而可能於大腸癌轉移的機制中扮演重要的角色。

The glycans on the surfaces of gastrointestinal tract-related cancer cells undergo remarkable changes during malignant transformation. The glycans specially expressing on the surfaces of cancer cells are called tumor-associated glycans. Sialyl Lewis a (sLea) and sialyl Lewis x (sLex) are the most famous tumor-associated glycans in gastrointestinal cancers. Previous studies indicated that sLea and sLex expression would promote the metastasis of gastrointestinal cancers and correlate with the risk of recurrence and the survival rate of a patient. sLea and sLex are constructed on the terminals of poly-LacNAc chains composed of repeated N-acetyllactosamine (Gal-GlcNAc，LacNAc) unit. In our previous research, we found that the expression of two glyco-genes, β-1,3-N-acetylglucosaminyltransferase 7 (B3GNT7) and I-branching β-1,6-N-acetylglucosaminyltransferase (IGnTA), which are involved in the biosynthesis of poly-LacNAc chains, are down-regulated in the tumor tissues of the patients with colon cancer. Therefore, the issues what we want to discuss are whether colon cancer cells would alter sLea and sLex expression and thereby affect colon cancer metastasis when the synthesis of poly-LacNAc chains on cell surfaces is altered. We used colon cancer cell lines as the cell models in our research to examine the effects of B3GNT7 and IGnTA expression on colon cancer metastasis and the mechanisms resulting in down-regulation of B3GNT7 and IGnTA in colon cancer cells. We found that sLea and sLex expression and the extent of colon cancer metastasis in colon cancer cells with over-expressing of B3GNT7 and IGnTA respectively are less than those of control cells. We also found that there are highly-methylated CpG islands in B3GNT7 and the 5’-regulated region of IGnTA in colon cancer cells. After treating colon cancer cells with the demethylating reagent, 5-Aza-2’-deoxycytidine (5AzadC), we found B3GNT7 and IGnTA expression are elevated and the extent of CpG islands methylation in B3GNT7 and the 5’-regulated region of IGnTA decreased. Therefore, the mechanisms regulating B3GNT7 and IGnTA expression in colon cancer cells are highly correlated with DNA methylation. Furthermore, we found that IGnTA expression increase and sLea expression decreases in colon cancer cells with treatments of epigenetic drugs, including 5AzadC and histone deacetylase inhibitors (HDAC inhibitors). In this research, we demonstrated that in colon cancer cells, B3GNT7 and IGnTA may play important roles in colon cancer metastasis and their expression pattern would affect sLea and sLex expression and DNA methylation may participate in the regulations of B3GNT7 and IGnTA expression.