鑑定具有改變Slit2剪接變異型表現的外來因子

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Title:	鑑定具有改變Slit2剪接變異型表現的外來因子 Identify extrinsic factors affecting the expression of Slit2 splicing form
Authors:	吳祥維 Wu, Siang-Wei
Contributors:	中山醫學大學:醫學研究所;蔡菁華
Keywords:	肺癌;醣蛋白 Slit2;splicing
Date:	2014
Issue Date:	2014-12-10T04:07:26Z (UTC)
Abstract:	Slit2是一個分泌型的醣蛋白，除了具有抑制神經軸突遷徙的能力外，也扮演著抑癌基因的角色。我們發現Slit2擁有兩種不同的剪接變異型(alternative splicing)，分別為具有exon15的Slit2-WT，以及缺乏exon15的Slit2-ΔE15。當Slit2-ΔE15在CL1-5細胞中過度表現時可以抑制細胞的侵犯(invasion)以及生長(growth)的能力；但是，Slit2-WT只具有抑制肺癌細胞的侵犯能力，而不影響生長。因此我們設計了一對可以區分Slit2-WT以及Slit2-ΔE15的引子(primer)，發現肺癌病人的腫瘤組織以及腫瘤周圍正常組織高度表現Slit2-WT，而Slit2-ΔE15表現很低或不表現。有趣的是，將近41%的氣胸病人(非癌症患者)，其肺組織之Slit2-ΔE15的表現量大於或等於Slit2-WT。除此之外，我們在人類正常支氣管上皮細胞(Beas-2B)中發現Slit2-ΔE15表現高於Slit2-WT，且在正常老鼠的肺組織中可以看到Slit2-WT以及Slit2-ΔE15皆有高度的表現。因此我們推測有兩種可能性，使得Slit2-ΔE15在肺癌病人中幾乎不表現。第一可能性是有部分個體的正常肺臟中表現較高之Slit2-WT，部分個體則是表現較高之Slit2-ΔE15，而表現Slit2-ΔE15很低的群體可能是罹患肺癌的高風險群；第二個可能性則是在癌化的過程可能會使腫瘤環境中Slit2的表現由Slit2-ΔE15轉變為以Slit2-WT為主，以達到創造利於癌細胞生長的環境。因此我們將肺癌細胞注射入老鼠的胸腔或者直接注射入老鼠的氣管內(IT)以測試這兩種可能性。但是這兩種動物模式無法成功的在肺組織中生長出腫瘤。未來我們將會建立一個肺部轉移的動物模式，以研究是否在腫瘤產生的過程中會改變Slit2選擇性剪接變異型的表現。我們對以Gallic acid、氯化鎳以及纖維母細胞生長因子FGF處理CL1-0肺癌細胞、人類胚胎肺纖維母細胞MRC5以及人類支氣管上皮細胞Beas-2B。結果顯示，處理gallic acid以及氯化鎳會增加CL1-0以及MRC5細胞的Slit2-ΔE15表現，而處理FGF則會增加MRC5細胞的Slit2-WT表現。這些結果顯示，Slit2在Exon15的選擇性剪接會受到外在環境的因子所調控。因此我們將會使用其他藥物，生長因子以及誘導發炎因子處理細胞，探討Slit2位在Exon 15上的選擇性剪接變異型表現之分子機制。找出在腫瘤微環境中參與調控Slit2-ΔE15轉變為Slit2-WT之分子機制，在未來將有助於控制肺癌的生長以及轉移。 Slit2 is a secreted glycoprotein, It plays important roles in axon guidance as well as in tumor suppression. We identified two alternative splicing forms of Slit2 at exon 15, Slit2-WT (presence of exon 15) and Slit2-ΔE15 (absence of exon 15). Slit2-ΔE15 possesses cell invasion and growth inhibition abilities; while Slit2-WT only inhibits cell invasion, without affecting cell growth. We generated a set of primers to differentiate Slit2-WT and Slit2-ΔE15 expression in cells. In lung cancer patients, the ratio of Slit2-ΔE15 to Slit2-WT expression in lung tumor and their normal counterpart pecimen was extremely low. Interestingly, nearly 41% of patients with pneumothorax, the expression of Slit2-ΔE15 were equal or higher than that of Slit2-WT in lung tissues. In addition, we found that Slit2-ΔE15 expression level was higher then Slit2-WT in normal human bronchial epithelial cells (Beas-2B), and both Slit2-WT and Slit2-ΔE15 had high expression levels in lung tissues of normal mice. Therefore, we raise two possibilities to explain why, Slit2-ΔE15 is alsmost not expressed in lung cancer specimens: first, it is possibile that some people express higher levels of Slit2-WT in normal lung tissue but some express higher level of Slit2-ΔE15. The Slit2-ΔE15 low expressers may be at higher risk for lung cancer; Secondly, during the process of carcinogenesis, the expression of Slit2 may be shifted from Slit2-ΔE15 to Slit2-WT in the tumor environment, creating an environment conducive to the growth of cancer cells. In order to test these possibilities, we injected lung cancer cells intratracheal (IT) or into the chest of mice. However, none of the models generated lung tumor successfully. We will establish lung metastasis models in the future to study whether there is alteration of Slit2 splicing druing tumorigenesis. We also treated CL1-0 lung cancer cells, MRC5 (human embryonic fibroblasts) and Beas-2B (Human bronchial epithelium) with gallic acid, NiCl2 and FGF. The results showed that gallic acid and NiCl2 induced Slit2-ΔE15 form expression in CL1-0 and MRC5 cells, while FGF enhanced Slit2-WT form expression. These results suggested that alternative splicing of Slit2 at Exon15 can be externaly regulated by environmental factors.Therefore, we will use other drugs, growth factors, inflammatory factors to treat cells for identifying mechanisms involved in alternative splicing of Slit2 at Exon15. Identifying molecules that can shift Slit-WT form expression to Slit2-ΔE15 form expression in tumor microenvironment would help to control the growth and metastasis of lung cancer.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/10087
Appears in Collections:	[醫學研究所] 博碩士論文

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