探討癌症基因體與表觀基因體的變化：從技術建立到臨床應用

Explore the Variety in Cancer Genome and Epigenome: from Technology Establishment to Clinical Application

張竣期(Chun-Chi Chang)

指導教授 ： 劉大智

共同指導教授 ： 張建國(Jan-Gowth Chang)

高雄醫學大學/醫學院/臨床醫學研究所/博士(2018年)

摘要

(一) 長的非編碼RNA（long noncoding RNA；lncRNA）具有多種功能，包括染色質的重塑與在轉錄和轉錄後對基因表現的調節。然而，只有少數的lncRNA已經被全面地研究，其中大部分都未被表徵。在本研究中，我們透過一個生物資訊學的管道，找出與蛋白質編碼基因3'非轉譯區（3'-UTRs）類似的新lncRNA序列。這些lncRNA和編碼基因對含有相同的miRNA靶位點，其中lncRNA CR933609與INO80D mRNA的3'-UTR匹配。CR933609和INO80D在非小細胞肺癌（NSCLC）和其他癌症組織中的表現量顯著降低。INO80D的表現量在CR933609敲低（knockdown）的NSCLC細胞中會降低，但CR933609的表現量在INO80D敲低的NSCLC細胞中不會降低。此外，我們證明了CR933609和INO80D各自有獨立的啟動子，還發現了在A549細胞中INO80D的表現量可由內源性miRNA-5096調降，但是不會在CR933609過度表現的A549細胞中被調降。因此，在NSCLC細胞中，CR933609可以充當誘餌以保護INO80D免受miRNA-5096的調降。我們建立了一個在3'-UTR中鑑定新型lncRNA的方法，並證明了它的存在。

(二) 細胞色素P450 2C8（CYP2C8）是負責花生四烯酸（arachidonic acid）與各種藥物代謝的主要酵素酶，並且會影響藥物之間的交互作用和一些相關疾病。不同種族間的CYP2C8酶活性與許多非同性質的遺傳變異性存在著大的個別化差異也都已經被報導了。因此，如何在不同人群中有效鑑定CYP2C8基因的多型性是非常重要的。高解析度熔點（high resolution melting；HRM）分析用於表徵CYP2C8基因的多型性。從95位正常台灣人的周邊血液樣品中抽取基因組DNA，通過HRM分析篩選CYP2C8基因的9個外顯子（exon），所有的結果均通過直接DNA定序確認。本研究中發現了五個新的單核苷酸多型性（single nucleotide polymorphism；SNP），在exon 8中有兩個SNP [1189G> A（D397N）和1230C> T（G410G）]，而其它三個SNP [1312G> C（E438Q），1497T> C（A499A）和1677delT（559delL）] 位於exon 9中。其中1497T> C（A499A）是最常見的變異，對偶基因頻率為20.53％，但並沒有改變合成後的氨基酸。即使CYP2C8和CYP2C9具有83％非常相似的cDNA序列，HRM分析仍然是一種快速、可靠、準確且經濟高效的基因突變篩檢方法。

關鍵字

長的非編碼核醣核酸 ； 3’端非轉譯區 ； 抑制誘餌 ； 高解析度熔點分析 ； 細胞色素Cytochrome P450 2C8 ； 基因多型性

並列摘要

(一) Long noncoding RNAs (lncRNAs) have various functions, including chromatin remodeling and the regulation of gene expression at both transcriptional and posttranscriptional levels. However, few lncRNAs have been comprehensively studied, with the majority being uncharacterized. In this study, we established a bioinformatics pipeline to identify novel lncRNA sequences similar to the 3′-untranslated regions (3′-UTRs) of protein-coding genes. These pairs of lncRNAs and coding genes contained the same miRNA target sites; the lncRNA CR933609 matched the 3′-UTR of INO80D mRNA. The expression levels of CR933609 and INO80D were significantly decreased in non-small-cell lung cancer (NSCLC) and other cancer tissues. The expression levels of both CR933609 and INO80D decreased in CR933609 knockdown NSCLC cells, but only INO80D expression levels decreased in INO80D knockdown cells. We proved that there are independent promoters in CR933609 and INO80D. We also found that expression levels of INO80D were downregulated by endogenous miRNA-5096 in A549 cells, but not in CR933609-overexpressing A549 cells. Furthermore, the lncRNA CR933609 acts as a decoy to protect INO80D from downregulation by miRNA-5096 in NSCLC cells. We established a protocol to identify novel lncRNAs in the 3′-UTR and proved the existence of novel lncRNAs.

(二) Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of arachidonic acid and various drugs and influences drug-drug interactions and some associated diseases. Large interindividual differences in CYP2C8 enzymatic activity and several nonsynonymous genetic variations have been reported in different races. Therefore, how to identify CYP2C8 polymorphisms efficiently for genotyping in different populations is very important. A high resolution melting (HRM) analysis was used to characterize the CYP2C8 polymorphism. Genomic DNA was extracted from peripheral blood samples from 95 normal individuals in Taiwan. Nine exons of the CYP2C8 gene were screened by HRM analysis. All results were confirmed by direct DNA sequencing. Five new single nucleotide polymorphisms (SNPs) were found in this study; two SNPs [1189G>A (D397N) and 1230C>T (G410G)] were in exon 8 and the others [1312G>C (E438Q), 1497T>C (A499A) and 1677delT (559delL)] were in exon 9. The 1497T>C (A499A) was the most common variant with an allele frequency of 20.53% but without amino acid substitution. HRM analysis is a fast, reliable, accurate and cost-effective screening method for gene mutations, even very similar cDNA sequences with 83% identities, compared with CYP2C8 and CYP2C9.

並列關鍵字

long non‑coding RNA ； 3'-untranslated region ； repressor decoy ； high resolution melting analysis ； CYP2C8 ； polymorphism

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