以核酸微陣列篩選與大腸直腸癌轉移相關的基因及訊息傳遞徑路

Using cDNA Microarray to Identify Candidate Genes and Signaling Pathways for Metastasis of Colorectal Cancer

陳興保(Hsin-Pao Chen)

指導教授 ： 陳世杰

高雄醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

前言：癌症的侵犯及轉移，是癌症致死的主因。癌症為基因變異所造成的疾病，它是由致癌基因，腫瘤抑制基因及其他修飾輔助基因的變異，使細胞原本正常運作調控的許多機制發生失控，而成為不受控制的癌細胞。許多基因及其相關的訊息傳遞徑路己逐漸被証實和癌細胞的生長、分化、細胞凋亡、血管新生、不停止的複製有關，但癌細胞的轉移則尚待進一步的研究與了解。所以，我們希望藉由核酸微陣列晶片的方法，來篩選出和大腸直腸癌轉移相關的基因及可能的訊息傳遞徑路，以做為進一步研究與臨床上追蹤治療的依据。
方法：我們採集6個早期大腸直腸癌及6個轉移性大腸直腸癌病人的檢體，以729個和癌症相關的基因，或癌細胞株選殖的核酸表現序列片段，做成核酸微陣列晶片，來測定這些病人的基因表現。以GeneSpring6.1軟體做資料分析，以基因表現差異2倍以上的方法，篩選出和大腸直腸癌轉移相關的基因，再用即時聚合酵素鏈反應的方法，確認這些基因表現結果是否和微陣列的結果是否一致，以了解微陣列晶片的可信度及結果，經這兩種方法篩選所得基因表現差異在2倍以上的基因，視為與大腸直腸癌轉移相關的基因，最後進入CGAP資料庫查詢這些基因相關的生物資訊以了解可能牽涉的訊息傳遞徑路。
結果：在本實驗中，以表現量倍數變化的比較，有3個上升表現的基因(PI3K,osteonectin, MMP14)，2個下降表現的基因(MAPK3, KIT)被篩選為和大腸直腸癌轉移相關的基因，再經定量即時聚合酵素連鎖反應的實驗，所得基因表現上升或下降的傾向，和微陣列資料是一致的，但上升或下降表現的程度，則不一致，使MAPK3和KIT變成不是大腸直腸癌轉移特異性的基因。所以，PI3K、Osteonectin及MMP14，是本實驗最後確認為與大腸直腸癌轉移相關的基因。而這些基因從CGAP資料庫的查詢中，可得知和細胞存活訊息活化、細胞淍亡的抑制、細胞外基質蛋白質解離的訊息傳遞徑路有關。
結論：本研究結果顯示，轉移性大腸直腸癌細胞，除了須具備有破壞組織間隔，細胞外基質的能力，並同時提高癌細胞移動力之外，細胞存活或抗細胞淍亡的表現，也是大腸直腸癌惡性程度的指標。因此，我們認為有這些基因或相關訊息傳遞徑路過度表現的病人，應做更積極的追蹤與冶療。

關鍵字

轉移 ； 大腸直腸癌 ； 基因表現 ； 核酸微陣列

並列摘要

Purpose: Cancer is the leading cause of death in our country for years. And colorectal cancer occupied the third cause of cancer related deaths. Invasion and metastasis are the major cause of cancer death. Cancer is a genetic disorder in entity. Cancer is caused by the changes of oncogenes, tumor suppressor genes and some modified function genes. It is known that expression of genes and related signal transduction pathways are related to cancer cell growth, differentiation, apoptosis, angiogenesis, and limitless replication. But the mechanism of metastasis remains unknown. Our goals are to screen the metastasis related genes and signaling pathways of colorectal cancer by cDNA microarray, the resulted genes and pathways can be regarded as the index of metastatic potential.
Method: We collected the specimens from 6 early colorectal cancer patients and 6 metastastic colorectal cancer patients. Cancer related genes and expression sequence tags (729) cloned from cancer cell line were collected as our cDNA microarray probes. GeneSpring 6.1 was adapted as data analysis software. Genes are selected based on expression level more than 2 fold change. Then the specific metastasis related genes were derived from the results that updown genes selected from metastatic group minus the updown genes selected from early colorectal cancer group. The results were further tested by quantitative real-time PCR. And the possible related singnaling pathways were derived from the CGAP database.
Results: In our experiment, 3 up regulated genes (PI3K, osteonectin, MMP14) are selected as the specific metastasis genes after microarray selection and validated by quantitative real time PCR. And these genes play the important roles in the related pathways such as cell survival, apoptosis, proteolysis in extracellular matrix and cell motility.
Conclusion: Our results show that the capacities of proteolysis in extracellular matrix, cellular migration were required for metatastatsis, the ability of cell survival and inhibition of apoptosis were important, too. These genes and related pathways may be used as the indices of the metastatic potential and aggressive treatment.

並列關鍵字

gene expression ； metastasis ； cDNA microarray ； colorectal cancer

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