兩種肺癌細胞株經游離輻射誘發之基因體研究 與Jagged 1所扮演之角色

Genomic Study of Radiation-Induced Transcriptional Responses and the Role of Jagged 1 in Two Closely-Related Lung Cancer Cell Lines

康 瑜(Yu Kang)

指導教授 ： 莊曜宇

共同指導教授 ： 蔡孟勳(Mong-Hsun Tsai)

國立臺灣大學/電機資訊學院/電機工程學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02808

摘要

肺癌是全世界最普及、致死率最高的癌症。非小細胞肺腺癌佔肺癌病人的百分之八十，不論是否為轉移的腫瘤，經由放射治療後的五年存活率僅達百分之五。然而，關於肺癌細胞對輻射線的反應機制仍不清楚。本研究以具有不同遷移能力之人類肺腺癌細胞CL1-0和CL1-5作為離體的研究對象。本研究目的在於利用微陣列晶片探討造成兩株肺腺癌細胞對輻射線敏感度差異之成因以及Jagged 1 基因在此兩細胞株中，細胞敏感度差異之中扮演的角色。首先使用群落形成測試法來比較兩株細胞對於輻射線敏感度的差異，接著以流式細胞分析儀分析在輻射線照射過後，細胞在不同時間點的細胞週期分佈。同時，以微陣列晶片實驗來探討在輻射線照射過後，兩細胞株相關基因變化以及分子機制。並且，在微陣列晶片資料中發現，Jagged 1的表現量在兩細胞的差異極大，而此表現形式也以即時聚合連鎖反應以及西方墨點法得到驗證。為了更近一步探討Jagged 1 在輻射線敏感度差異中扮演的角色，在CL1-0中大量表現Jagged 1，並且進行功能測試。結果顯示，CL1-5 相較於CL1-0來說，對於輻射線具有較高的敏感性。經由10 Gy 加瑪射線照射之後，CL1-0和CL1-5 均在24小時的時間點，具有最高比率的細胞停滯在G2/M期，並且均缺乏G1/S期的滯留。將微陣列的資料經由密集叢聚分析結果可知，有六個集群在兩細胞株之中表現形式差異極大，這些集群中的基因和細胞死亡、細胞週期、細胞生長、增殖與細胞功能維持有關。在其中一個不同表現的集群中，具有和G2/M細胞檢查點相關的基因，與實驗中G2/M 細胞週期停滯之引發吻合。 在CL1-0中大量表現Jagged 1之功能測試結果中發現，相較於CL1-0，其經由10 Gy輻射線照射之後，存活率上升，而經由即時聚合連鎖反應可知，其細胞中NFκBIA 的含量改變，由流式細胞分析儀的結果可知，其在24 小時，sub G1 以及 G2/M 的細胞分佈比例介於CL1-0 與CL1-5之間。 综合上述，CL1-5較CL1-0來得具有輻射線敏感性，而CL1-5主要的死亡機制是透過增殖死亡，除此之外，Jagged 1 與CL1-0以及 CL1-5輻射敏感性差異有相關連，可能與NFκBIA具有交互作用，進而影響存活率。

關鍵字

肺癌 ； 輻射 ； 輻射敏感度 ； 微陣列晶片 ； Jagged 1

並列摘要

Lung cancer is the most common and lethal cancer in the world. NSCLC accounts for 80% lung cancer patients, and the 5-year survival rate after radiotherapy is about 5% whether the patients are with less or more extensive tumors. However, the mechanism behind radiation response of lung cancer is still not clear. Here, lung adenocarcinoma cell lines CL1-0 and CL1-5 with different metastasis abilities were used as the objects of our in vitro study. The aim of this thesis was to investigate the radiosensitivity in CL1-0 and CL1-5 via global gene expression profiles and the role of a notch ligand, Jagged 1 played in radiosensitivity. Clonogenic assay was used to obtain radio sensitivity in CL1-0 and CL1-5. To better understand the cell cycle distributions followed by radiation, flow cytometry experiment was performed at different time points. At the same time, the temporal global gene expression profile was obtained to understand the radiosenstivity via global transcriptional activities. In addition, the expression difference of Jagged 1 between CL1-0 and CL1-5 were significant in microarray, and were further confirmed by real time PCR and western blotting. To investigate the role of Jagged 1 in radiosensitivity, Jagged 1 overexpressed CL1-0 was obtained to perform functional assay. The data indicated that CL1-5 was more radiosensitive than CL1-0 after 10 Gy radiation. The irradiated CL1-0 and CL1-5 exhibited G2/M blockade and with the lack of G1/S checkpoint. Moreover, the subG1 area was significantly higher in the irradiated CL1-0 than the CL1-5 cells. From microarray data and through tight clustering analysis, there were six clusters expressed significantly different between CL1-0 and CL1-5. Genes among them were related to cell death, cell cycle, cell growth and proliferation, cellular function and maintenance. A different expression gene group between CL1-0 and CL1-5 showed genes related to G2/M checkpoint, which was consistent with the G2/M arrest activities. The Jagged 1 functional assay results showed an increasing survival rate in Jagged 1 overexpressed CL1-0 compared to CL1-0.Also, the transcription level of NFκBIA were changed in Jagged 1 overexpressed CL1-0. Compared to CL1-0 and CL1-5, Jagged 1 overexpressed CL1-0 cell line showed a mid percentage in sub G1 and G2/M phase. In conclusion, CL1-5 was more radio sensitive than CL1-0 and the death cause was mainly from mitotic death. In addition, Jagged 1 was associated partially with the radiosenstivity in CL1-0 and CL1-5, and may have a cross talk with NFκBIA.

並列關鍵字

lung cancer ； radiation ； radiosensitivity ； microarray ； Jagged 1

參考文獻

1. Parkin, D.M., et al., Global cancer statistics, 2002. CA Cancer J Clin, 2005. 55(2): p. 74-108.

2. Jemal, A., et al., Cancer statistics, 2007. CA Cancer J Clin, 2007. 57(1): p. 43-66.

3. 行政院衛生署衛生統計資料網, Health and National Health Insurance Annual Statistics Information Service

4. Travis, W.D., Pathology of lung cancer. Clin Chest Med, 2002. 23(1): p. 65-81, viii.

5. Spira, A. and D.S. Ettinger, Multidisciplinary management of lung cancer. N Engl J Med, 2004. 350(4): p. 379-92.