探討Shisa3基因於大腸直腸癌之基因靜默機制 及其臨床相關性

Abstract

大腸直腸癌 (Colorectal cancer，CRC)自正常大腸黏膜細胞轉變成癌細胞乃是經由長時間累積基因變異所造成。而大腸直腸癌致癌機轉分成基因遺傳 (Genetic)及表觀基因 (Epigenetic)調控之路徑。本論文研究的Shisa3基因位於人類第四號染色體4p13，相較於鄰近正常黏膜組織，CRC腫瘤組織Shisa3基因mRNA的表現明顯下降，推測Shisa對於CRC可能具有相關的抑癌功能。至今，人類Shisa3基因之功能及其在癌症發展中所扮演的角色尚不清楚，因此，本論文主要研究目的是探討Shisa3基因表現於CRC中受抑制的機制及其臨床的相關性。首先，為確認Shisa3基因的表現量，我們利用RT-PCR及qRT-PCR檢測其在不同CRC細胞株及臨床檢體的mRNA表現，結果顯示：大部分癌細胞株及臨床腫瘤檢體中，Shisa3基因都表現下降。接著探討Shisa3基因的靜默機制 (silencing)，利用DNA套數分析及對偶基因失異合性的初步研究，結果顯示基因刪除不是抑制Shisa3基因表現的主要機制。於表觀基因調控之研究，我們利用DNA去甲基化藥物5-Aza-2’-deoxycytidine (5-aza-CdR)處理六株大腸直腸癌細胞株，發現五株細胞(83.3%)可明顯恢復Shisa3基因表現。同時，Shisa3基因自Promoter區域至Exon1間具有高密度的CpG位點分布，因此，我們利用亞硫酸鹽定序聚合酶連鎖反應 (bisulfate-sequencing PCR，BSP)檢測CRC細胞株及臨床檢體於Shisa3基因CpG高分布區內甲基化的情形，並找出最有可能調控此基因表現的CpG區域。接著針對特定CpG位點設計MethyLight和Pyrosequencing兩種方法確認Shisa3基因的CpG調控區並定量DNA甲基化程度。結果顯示：於CRC腫瘤之DNA甲基化程度明顯高於鄰近正常黏膜組織，同時DNA甲基化程度與Shisa3基因表現量具有負相關 (R2=0.3248, p<0.0001)。進行臨床相關性分析，發現：Shisa3基因發生高度甲基化與CRC疾病復發有顯著相關，同時病人之總存活率 (Overall survival, OS)和無疾病存活率 (Disease-free survival, DFS)亦明顯降低。因此， Shisa3基因高度甲基化可能做為CRC病患之疾病復發及存活率的預後分子標記。

Most colorectal cancer (CRC) results from a consequence of the accumulation of genetic alterations that transform normal colonic mucosa into adenocarcinoma. Previous reports indicated that pathogenesis of CRC involves two different mechanisms, namely genetic and epigenetic pathways. Shisa3 gene, which we focused on, locates on human chromosome 4p13. Compared with matched normal colon mucosa, the mRNA expression of Shisa3 gene was decreased in most CRC primary tumors. Accordingly, we propose that Shisa3 might have tumor suppressor function in CRC. Nowadays the role of Shisa3 in tumorigenesis is still unknown. Therefore, the aim of this study is to investigate Shisa3 gene silencing and it’s clinical correlation in CRC. First, we used CRC cell lines and primary tissues to determine mRNA expression of Shisa3 gene by RT-PCR and qRT-PCR. The results showed that down-regulation of Shisa3 in most CRC cell lines and primary tumors. To ascertain whether Shisa3 gene is silenced via genetic alterations in CRC, we performed Shisa3 DNA copy number and loss of heterozygosity (LOH) analyse. Our data showed that genetic deletion isn’t the major mechanism of Shisa3 gene silencing. Meanwhile, we found that Shisa3 expression in 5 of 6 (83.3%) CRC cell was restored after treatment with 5-aza-2’-deoxycytidine (5-Aza-CdR). Besides, high density CpG islands span from promoter to Exon 1 of Shisa3 gene. Accordingly, we analyzed DNA methylation status of Shisa3 at various CpG sites in CRC cell lines and primary CRC tissues by bisulfite-sequencing PCR (BSP). To investigate the possible regulation sites of DNA methylation in Shisa3, the DNA methylation level of Shisa3 was determined by MethyLight and pyrosequencing methods. The reverse correlation was observed between DNA methylation and mRNA expression of Shisa3 gene in CRC tumors (R2=0.3248, p<0.0001). Moreover, we revealed the impact of Shisa3 DNA methylation on patients’ overall survival and disease-free survival. In conclusion, the methylation level of Shisa3 gene might be a possible prognostic biomarker of CRC in the future.