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標題: | 探討osteopontin在神經膠瘤細胞對HIF-1α的調節作用 Regulation of HIF-1α by osteopontin in glioblastoma cells |
作者: | Peng-Ru Chen 陳鵬如 |
指導教授: | 符文美 |
關鍵字: | 骨調素,缺氧,神經膠瘤,組合蛋白,去組合蛋白, osteopontin,hypoxia,glioma,integrin,disintegrin, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 人類惡性神經膠瘤,是最常見的原位性腦瘤,其特性是廣泛的浸潤到鄰近的正常腦組織。目前用來治療神經膠瘤的方法包含手術治療、傳統放射治療、或是化學藥物治療,但是都因為這項特徵而使得治療的效果有限。骨調素(osteopontin)是一種磷酸化的醣蛋白,一些癌細胞會過度分泌骨調素,而大量的骨調素與癌症的惡化有關。有文獻指出骨調素參與癌症的進程,例如異常的細胞分裂、細胞遷移、細胞侵入、和血管新生。除此之外,也有文獻指出在缺氧的情況下觀察到骨調素的表現增加。癌細胞在缺氧的情況下會藉由穩定缺氧誘導因子(HIF)來克服缺氧時所面對的壓力。基於以上的觀點,我們探討在神經膠瘤細胞中骨調素對HIF-1α表現的調控作用,並且探討disintegrin (HSA(C34S)-ARLDDL、PEG-ARLDDL)的抑制作用,這些disintegrins是從Rhodostomin中改變RGD domain附近胺基酸序列所獲得的衍生物,對αvβ3有專一性的拮抗作用。在本篇研究結果顯示人類神經膠瘤細胞U87在缺氧的情況下會促進骨調素的表現。外給骨調素會促使在缺氧下累積更多的HIF-1α蛋白,但不影響HIF-1αmRNA的表現,而osteopontin增加蛋白之表現會被disintegrin HSA(C34S)-ARLDDL所拮抗。此外,利用西方點墨法分析核內蛋白及免疫螢光染色證實骨調素會促進HIF-1α蛋白轉移到核內。更進一步發現,外給骨調素可以促進U87在缺氧下分泌更多的VEGF以及更嚴重的細胞侵入(invasion),但是在給予disintegrin PEG-ARLDDL後便會抑制此U87的侵入行為。另外我們轉殖一小段具有專一性拮抗骨調素mRNA的RNA,可使骨調素的表現量下降。骨調素表現缺乏的U87細胞在CoCl2的刺激之下比起正常的U87細胞表現較少的HIF-1α蛋白,利用HRE-luciferase reporter assay也發現此種細胞有較少的HIF-1α轉譯活性。在缺氧的情況下骨調素缺乏的U87細胞比起正常的U87細胞有較少的細胞遷移 (migration)。此外,在骨調素缺乏的U87細胞中給予骨調素會恢復其HIF-1α的表現量,而此現象會被LY294002(PI3K的抑制劑)及SB203580(p38的抑制劑)所拮抗,進一步我們也觀察到外給骨調素會增加這些細胞中Akt、p38及FAK的磷酸化。
總結本篇的研究結果,我們發現在人類神經膠瘤細胞中給予骨調素會促進缺氧下所誘導的HIF-1α蛋白的堆積及細胞的侵入,在給予disintegrin後便可拮抗此作用。此外我們也證實了人類神經膠瘤細胞在缺氧的環境下骨調素與HIF-1α蛋白之間的關係。骨調素可能是藉由integrin、PI3K及p38這些訊息傳遞路徑來調控HIF-1α蛋白的表現。 Human malignant gliomas, the most common primary brain tumors, are characterized by their ability to infiltrate to adjacent normal brain tissue. This infiltrative feature causes the limit effectiveness for present therapies including surgery, radiation therapy and chemotherapy. Osteopontin, a phosphorylated glycoprotein, is overexpressed in several cancers and correlates with tumor grade. Previous reports have indicated that osteopontin participates in tumor progression such as proliferation, migration, invasion, and angiogenesis. In addition, elevated osteopontin expression is observed following hypoxia, and tumor cells overcome hypoxia stress by the stabilization of hypoxia inducible factor (HIF) transcriptional regulators. Based on these viewpoints, we investigated the effect of osteopontin on HIF-1α expression in glioblastoma, and explored the inhibitory effects of two kinds of αvβ3-selective disintegrins (HSA(C34S)-ARLDDL, PEG-ARLDDL) derived from the mutation of RGD neighborhood of Rhodostomin. It was found that the expression of osteopontin increased after exposure to hypoxia in U87 human glioma cells. Exogenous application of osteopontin enhanced hypoxia-induced HIF-1α protein expression, which was attenuated by the co-treatment with disintegrin HSA (C34S)-ARLDDL. Osteopontin at concentration of 3 ng/ml did not affect HIF-1α mRNA expression. In addition, we showed that application of osteopontin facilitated HIF-1α translocation to nucleus using nuclear extract Western blot analysis and also confirmed by the nuclear localization of HIF-1α using immunofluorescent staining. Furthermore, it was found that exogenous osteopontin enhanced hyoxia-induced VEGF secretion and facilitated U87 cell invasion, which was antagonized by the disintegrin of PEG-ARLDDL. We further transfected U87 MG with a specific small hairpin RNA to knockdown osteopontin expression. Osteopontin-deficient U87 MG cells gave rise to less HIF-1α protein than control cells, and less HIF-1α transcriptional activity estimated by HRE-luciferase reporter assay after CoCl2 treatment. Osteopontin knockdown decreased the U87 MG cell migration under hypoxia. Furthermore, application of osteopontin to osteopontin-knockdown cells could reverse the HIF-1α expression, which was attenuated by the treatment of LY294002 (PI3K inhibitor) and SB203580 (p38 inhibitor). Moreover, exogenous osteopontin also increased the phosphorylation level of Akt, p38, and FAK. In conclusion, osteopontin enhanced hypoxia-induced HIF-1α protein accumulation and invasion in human U87 glioma cells. Treatment of disintegrin could antagonize these effects. Moreover, a relationship between osteopontin and HIF-1α in glioblastoma cells under hypoxia treatment was demonstrated. The regulation of osteopontin on HIF-1α protein may act through integrin,PI3K, and p38 signaling pathways. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40798 |
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