摘要

腦膠質細胞瘤是原發性腦腫瘤中最常見的類型，為人類癌症中具高度侵略性及侵襲性之神經腫瘤。介白素33 (IL-33) 被認為是一種具雙功能之細胞因子，在組織損傷時能通過酵素促進細胞裂解，進而充當細胞因子之警報蛋白；並且其 N 端包含染色質結合基序及核定位序列 (NLS)，可調節細胞核之位置轉換並與異染色質中之組蛋白結合。實驗室先前研究表明，IL-33 可促進腦膠質瘤之致瘤性及影響微小膠細胞之遷移，但對於細胞核內 IL-33之功能仍然不甚瞭解。在本研究中，利用人類神經膠質瘤細胞株 (U251MG 及 U87MG) 為平台，並使用慢病毒轉染調節 IL-33 表現量 (IL33KD 及 IL33oe)，進一步了解 IL33 於細胞核中之生物功能。本研究發現，IL-33 高度表達之腦膠質瘤細胞 (IL33oe) 對於 DNA 損傷藥物—TMZ的作用具有抵抗力，可能與 IL33oe 細胞中與 DNA 修復相關之基因 (BRCA1、BRCA2、Brcc3、Rad51、FANCB 以及 FANCD）表達顯著增加有關。利用穿透式電子顯微鏡 (TEM) 以及組蛋白 H2A 之免疫螢光染色分析腦膠質瘤細胞核之形狀，IL33KD 減弱了異常具癌變性之細胞核特徵，包括細胞核之凹槽及裂縫情形，並縮小甜甜圈狀孔洞；反之，IL33oe 加劇了癌變之核凹槽及裂縫，使甜甜圈狀孔洞變大，並出現多葉狀結構之細胞核異常情形。進一步利用西方墨點法分析組蛋白 (H2A及H3) 之表現量；IL33KD 使其表現量顯著降低，而IL33oe 則是顯著增加。另外在非組蛋白之 DNA 結合蛋白，HMGA1 及 HMGA2 也觀察到相同之結果。綜合以上，細胞核內 IL-33 之高度表達伴隨 DNA 修復相關之基因表達增加，進而導致腦膠質瘤細胞對於 DNA 損傷藥物產生抵抗性。並且IL-33 核蛋白會與染色質相關蛋白相結合，進而調節腦膠質細胞瘤之細胞核結構，從而導致癌細胞之擴展及惡化。

SUMMARY

Glioma, the most common subtype of primary brain tumor, is aggressive and highly invasive neurologically tumor. Interleukin-33 (IL-33) belongs to IL-1 family and functions as an alarmin upon tissue damage via the interaction with its receptor ST2 at the extracellular space after the cleavage with proteinase. However, the full length of IL-33 protein contains N-terminal homeodomain-like helix-turn-helix that includes a chromatin-binding motif and nuclear localization sequence (NLS). Thus, IL-33 is referred as a nuclear protein to have potential in the regulation of nuclear translocation and association with histones in heterochromatin. The previous study from our laboratory has shown that IL-33 might promote glioma expansion and microglia migration via the interaction with ST2. Given the fact that IL-33 is expressed abundantly in the glioma cell nucleus, its nuclear function remains to be defined in depth. In this study, using lentivirus-mediated IL33 gene knockdown (IL33KD) and IL-33 overexpression (IL33oe) in human glioma cell lines (U251MG and U87MG), we found that IL33oe-glioma cells had resistance to the insults of temozolomide (TMZ), possibly because of the increased expression of DNA repair genes (BRCA1, BRCA2, Brcc3, Rad51, FANCB, and FANCD) in IL33oe-glioma cells. Next, examination of glioma nuclear shape from transmission electron microscopy (TEM) analysis and immunofluorescence for histone protein H2A staining. The results show that IL33KD attenuated the abnormal cancerous nuclear characteristic including indentation, long clefts, and doughnut-like shape. whereas these, IL33oe promoted the changes in glioma nuclear shapes, such as the formation of multiple lobes. Moreover, IL33KD decreased nuclear H2A and H3 levels and high mobility group protein 1/2 (HMGA1/2) in U251MG. In contrast, forced overexpression of IL-33 in U251MG (IL33 oe U251MG) increased nuclear H2A/H3 levels and HMGA1/2. Furthermore, the results through the pull-down analysis showed the interaction of H2A and IL-33 in glioma cells. Altogether, the upregulation of nuclear IL-33 expression was along with an increase in DNA repair-associated gene expression, contributing to desensitization of glioma cells to DNA damaging agent. Moreover, nuclear IL-33 proteins in cooperation with chromatin-associated proteins regulates glioma nuclear structure, which might be crucial for glioma progression and malignancy.

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