- 學位論文
非編碼核醣核酸在癌症分子生物學的調控機轉及臨床應用研究
The regulation mechanism and clinical application of noncoding RNAs in the molecular biology of human cancers
摘要
研究背景：長非編碼核醣核酸(Long noncoding RNAs, lncRNAs)在細胞癌化扮演重要的角色。 LncRNA taurine-upregulated gene 1 (TUG1) 被證實與一些人類癌症相關。但是TUG1-involved regulation 的機轉在文獻中很少被提到，因此，與TUG1調控的相關轉仍有許多未知的情形。 研究方法：我們檢測在89位患有非小細胞肺癌( non-small cell lung cancer ,NSCLC)的病患檢體中檢測TUG1表現的情形，以及分析與臨床特徵的相關性。我們使用circular chromosome conformation capture (4C) coupled with next-generation sequencing 來探查基因體中與TUG1有相互作用的序列。 研究結果： TUG1的表現量在腫瘤組織中有明顯downregulated，並且與性別(p = 0.006)、抽菸狀態(p = 0.016)及腫瘤分化程度(p = 0.001)有明顯相關。TUG1 knockdown 會促進NSCLC 細胞的生長情形。根據TUG1 4C sequencing data 生物資訊的分析結果83個基因被辨認出來。這些候選基因當中CUGBP and Elav-like family member 1 (CELF1) 有可能是 TUG1 in-trans調控的標的。為了確認TUG1 和 CELF1之間相關調控，我們檢測TUG1 knockdown H520 cells的CELF1表現量。結果顯示CELF1表現量在TUG1 knockdown H520 細胞中顯著上升。核醣核酸免疫沉降實驗顯示TUG1 RNA 和 EZH2/EED (PRC2複合物)相連結。染色質免疫沉澱技術實驗進一步顯示 EZH2/EED 與CELF1 啟動子區在the transcript start site上游992 bp近處有結合。 結論： TUG1 在非小細胞肺癌有downregulated的情形。使用 TUG1 4C sequencing和bioinformatic analysis，我們發現 CELF1可能是TUG1 RNA in-trans regulation的一個作用標的。接續的實驗證實TUG1/PRC2/CELF1的調控路徑。這個新發現的調控路徑可能作為了解非小細胞肺癌生成與疾病治療的關係。
並列摘要
Background: Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, and lncRNA taurine-upregulated gene 1 (TUG1) has been shown to be associated with several human cancers. However, the mechanisms of TUG1-involved regulation remain largely unknown. Methods: We examined the expressions of TUG1 in a cohort of 89 patients with non-small cell lung cancer (NSCLC) to determine the association between TUG1 expression and clinical parameters. We used circular chromosome conformation capture (4C) coupled with next-generation sequencing to explore the genome regions that interact with TUG1 and the TUG1-mediated regulation. Results: TUG1 was significantly downregulated, and the TUG1 downregulation correlated with sex (p = 0.006), smoking status (p = 0.016), and tumor differentiation grade (p = 0.001). Knockdown of TUG1 significantly promoted the proliferation of NSCLC cells. According to the bioinformatic analysis result of TUG1 4C sequencing data, 83 candidate genes and their interaction regions were identified. Among these candidate genes, CUGBP and Elav-like family member 1 (CELF1) are potential targets of TUG1 in-trans regulation. To confirm the interaction between TUG1 and CELF1, relative expressions of CELF1 were examined in TUG1 knockdown H520 cells; results showed that CELF1 was significantly upregulated in TUG1 knockdown H520 cells. RNA immunoprecipitation was then performed to examine whether TUG1 RNA was bound to PRC2, a TUG1-involved regulation mechanism reported in previous studies. The results indicated that TUG1 RNA was bound to enhancer of zeste protein 2/embryonic ectoderm development (EZH2/EED), which is essential for PRC2. Finally, our designed ChIP assay revealed that the EZH2/EED was bound to the promotor region of CELF1 within 992 bp upstream of the transcript start site. Conclusion: TUG1 is downregulated in NSCLC. Using TUG1 4C sequencing and bioinformatic analysis, we found CELF1 as a potential target of TUG1 RNA in in-trans regulation. Moreover, subsequent experiments showed that TUG1 RNA could bind to PRC2 in the promotor region of CELF1 and negatively regulate CELF1 expressions in H520 cells. Our results may facilitate developing new treatment modalities targeting TUG1/PRC2/CELF1 interactions in patients with NSCLC.