辨認在肺腺癌中受到不正常DNA甲基化調控之基因

Abstract

肺腺癌好發於非吸菸的亞洲女性，屬於肺癌分類下的非小細胞肺癌。在西方國家與台灣中肺癌都具有很高的致死率，而早期診斷的困難和缺乏有效的治療方法都可能是造成高死亡率的原因。近年來，不正常的DNA甲基化可以調控基因表現並且被證實與癌症相關，因此常常被視為診斷的標記與藥物發展的目標。藥物處理可以改變DNA甲基化程度，進而影響基因的表現，以達到治療的目的。 然而，在台灣肺腺癌的研究中，對於受甲基化調控的基因與其功能還不清楚。因此，我們搜集台灣非吸菸女性的肺腺癌檢體以及對應的周邊正常組織，以微陣列晶片(microarray)檢測基因表現與DNA甲基化程度，並且將這兩個微陣列晶片的資料整合，分析出肺腺癌中可能受到甲基化調控的基因。分析結果得到167個基因，其中108個基因屬於高甲基化、低基因表現；另外59個基因屬於低甲基化、高基因表現。經過進一步篩選與比對IPA，得出六個目標基因進行驗證。以即時定量聚合連鎖反應(qPCR)檢驗基因表現，並以亞硫酸鹽定序(bisulfite sequencing)與Sequenom檢驗基因DNA甲基化程度。本篇研究概略呈現了肺腺癌中全基因組甲基化程度與基因表現圖譜的關係，以及篩選出一群可能受到DNA甲基化調控的基因。

Lung adenocarcinoma, especially often observed in Asian women and never-smokers, was the most prevalent histological subtype of non-small cell lung cancer (NSCLC), which accounted for the majority of lung cancer. Lung cancer was in high mortality either in western countries or in Taiwan. The high mortality of lung cancer was contributed to the difficulty of early diagnosis and the lack of effective therapies. Since aberrant DNA methylation was mostly involved in cancers and was reversible, the methylation-regulated genes were taken as diagnosis markers or potential targets for drug development. However, the methylation-regulated genes and their functions in lung adenocarcinoma in Taiwan were still unclear. To investigate gene expression and DNA methylation levels in lung adenocarcinoma, the tumor and adjacent normal tissues of non-smoking Taiwanese female were collected. Microarray data of expression and methylation were analyzed concurrently to find out the methylation-regulated genes. Selection criteria was set according to significant expression differences between tumor and normal tissues, and the negative correlation between expression profiles and methylation patterns. One hundred and eight genes were hypermethylated and down-regulated; 59 genes were hypomethylated and up-regulated. After additional strict criteria and Ingenuity Pathways Analysis, six candidate genes were validated both in gene expression by qPCR and DNA methylation levels by bisulfite sequencing and Sequenom either in lung adenocarcinoma specimens or lung cancer cell lines. In summary, our study sketched the genome-wide methylation patterns and expression profiling in lung adenocarcinoma, and identified a group of candidate genes regulated by DNA methylation.