基因體甲基化圖譜與抑癌基因甲基化參與肺癌形成之機制及臨床應用探討

Abstract

癌症一般認為與基因體與外顯基因體發生變異有關，而在外顯基因體研究中，基因啟動子過度甲基化是最主要造成基因不活化的原因之一。抑癌基因的啟動子過度甲基化，會造成抑癌基因不活化，進而導致癌症的發生。為了鑑定在癌症基因體中，過度甲基化的區域所包含的可能新穎抑癌基因，本研究利用差異甲基化雜交法（differential methylation hybridization）的微陣列分析及染色質免疫沈澱晶片分析法（chromatin immunoprecipitation -on -chip），針對30位非小細胞肺癌病人及數個肺癌細胞株進行基因體的過度甲基化區域及染色質鬆緊狀態研究。結果發現在不同的肺癌子類型及肺癌分期，有特定的基因被過度甲基化，這些過度甲基化基因也許可以作為早期偵測及預測癌症發展的生物指標。 此外，在肺癌病人的差異甲基化雜交法的結果中，本研究發現一個與抗細胞增生、細胞靜止與細胞分化的COL14A1基因啟動子有過度甲基化的情形，而且在染色質免疫沈澱晶片分析法中，COL14A1啟動子的染色質區域相較於正常肺細胞，肺癌細胞呈現較為緊密的狀態。此外，本研究發現有60.4%的非小細胞肺癌病人有COL14A1基因啟動子過度甲基化的情形，而且其mRNA及蛋白質分別有50.0%及43.9%的低表達情形；另外本研究也發現COL14A1基因啟動子過度甲基化與晚期肺癌病人有統計相關。這些驗證實驗顯示外顯基因體研究是尋找癌症相關基因的有效工具，COL14A1基因及其蛋白變異參與肺癌的分子機制將進一步由細胞及動物模式研究來鑑定。 在本實驗室先前對基因體缺失的研究中，發現在染色體3p21的區域有高達50%以上的基因座缺失情形。此外，在差異甲基化雜交法的結果中，也發現位於染色體3p21.3的RASSF1基因在肺癌早期的病人中有過度甲基化情形，因此染色體3p21.3區域的基因不活化對於台灣地區肺癌形成扮演一個非常重要的角色。而RASSF1A及BLU這二個頭尾相連的抑癌基因位於染色體3p21.3的區域，由於這二個基因位置非常靠近，因此本研究預測這二個基因的表達及啟動子過度甲基化具有區域效應，也就是此二基因的表達及基因甲基化具有一致性。如果沒有區域效應，可能是因為RASSF1A及BLU基因之間具有絕緣子（insulator）構造所導致。首先，本研究針對32位肺癌病人，利用特定序列甲基化微陣列分析法（methylation- specific oligonucleotide microarray）及反轉錄聚合連鎖反應，找出會影響RASSF1A及BLU基因mRNA表達的關鍵轉錄CpG位置。同時也發現在RASSF1A基因的關鍵轉錄CpG位置上，有E2F1這個轉錄因子的結合，當這些位置被過度甲基化時，會使E2F1無法結合在RASSF1A的啟動子上，導致RASSF1A基因表達下降。此外，本研究發現RASSF1A及BLU這二個基因各自的關鍵轉錄 CpG位置的甲基化與各自基因的低轉錄與低轉譯有關；然而，這二個基因的甲基化狀態及基因表達卻沒有一致性，也就是沒有區域效應。利用免疫沈澱聚合連鎖反應（chromatin immunoprecipitation-PCR）證明CTCF蛋白結合在RASSF1A及BLU基因啟動子之間的絕緣子上，也利用亞硫酸鹽定序（bisulfite sequencing）發現在絕緣子兩端的甲基化不連續情形。所以CTCF也許提供了屏障效應導致這二個基因沒有所謂的區域效應。本研究找出了RASSF1A及BLU的關鍵轉錄CpG位置，這些位置的甲基化會影響基因的表達；同時也證明了CTCF結合在RASSF1A及BLU之間，使得這二個基因的表達沒有區域效應。本研究為首篇鑑定影響RASSF1A及BLU基因mRNA表達的關鍵轉錄CpG位置的報導，並提出絕緣子可以做為如染色體3p21基因群座（gene cluster）屏障效應的證據。

Cancer is caused by the accumulation of both genetic and epigenetic changes. Promoter hypermethylation is one of the major epigenetic changes that cause gene inactivation. Aberrant promoter hypermethylation of CpG islands associated with tumor suppressor genes (TSGs) can lead to transcriptional silencing and result in tumorigenesis. The genomic regions with hypermethylation status may possess novel candidate TSGs. The present study used a microarray-based epigenome-wide methylation analysis called differential methylation hybridization (DMH) to identify the regions of hypermethylation and a chromatin immunoprecipitation (ChIP)-on-chip analysis to identify the regions of condensed or open chromatin in 30 non-small cell lung cancer (NSCLC) patients and several lung cell lines, and have successfully detected several cancer subtype- and stage-specific hypermethylated genes. They may serve as biomarkers for the early detection or prognosis prediction of lung cancer. Using DMH, this study identified promoter hypermethylation of the COL14A1 (collagen, type XIV, alpha 1) gene, which has cell anti-proliferative activity and plays a role in cell quiescence and differentiation. Using ChIP-on-chip, COL14A1 promoter region was shown to be in compact chromatin structure in cancer cell lines compared to normal cell line. In addition, 60.4% of 48 NSCLC patients showed COL14A1 promoter hypermethylation and coincided with low mRNA and protein expression. Moreover, COL14A1 promoter hypermethylation was significantly associated with late stage lung cancer patients. The present study provided evidence that epigenomic tools such as DMH and ChIP-on-chip can be used for identifyication of cancer-related genes such as COL14A1. In previous study of my laboratory, there was more than 50% of loss of heterozygosity in chromosome 3p21. In addition, RASSF1 promoter hypermethylation in chromosome 3p21.3 was shown in early stage patients of NSCLC in DMH data. Therefore, gene silencing in chromosome 3p21.3 is important for lung tumorigenesis in Taiwan. Tumor suppressor genes RASSF1A and BLU are two tandem head-to-tail genes located at 3p21.3. The current study hypothesized that there may be a regional effect on their gene expression and promoter methylation status. If not, then there may be an insulator between RASSF1A and BLU genes. This study first identified transcriptionally important CpG sites using the methylation-specific oligonucleotide microarray in relation to mRNA expression of RASSF1A and BLU genes in primary lung tumors. The data demonstrated that E2F1 bound to the transcriptionally important CpG sites in RASSF1A gene, and this transcriptional regulation was impaired when the targeted CpGs were hypermethylated. Both RASSF1A and BLU genes had their own transcriptionally important CpG regions. However, there was no correlation of methylation status between RASSF1A and BLU. Using chromatin immunoprecipitation-PCR (ChIP-PCR), CCCTC-binding factor (CTCF) was found to bind to insulator sequences located between these two genes. Bisulfite sequencing and ChIP-PCR revealed distinct methylation and chromatin boundaries separated by the CTCF binding domains. This study dissects for the first time the transcriptionally important CpG sites for both RASSF1A and BLU genes and demonstrates that CTCF binding to the insulator of BLU gene possesses a barrier activity within separate epigenetic domains of the juxtaposed BLU and RASSF1A loci in the 3p21.3 gene cluster region.