砷甲基化及p14、p15和p16基因異常與上泌尿道泌尿上皮癌之相關性

The Relationships among Arsenic Methylation, Gene Alterations of p14, p15, and p16 and Upper Urinary Tract Urothelial Carcinoma

林建維(Chien-Wei Lin)

指導教授 ： 薛玉梅

共同指導教授 ： 張淑芬

臺北醫學大學/公共衛生暨營養學院/公共衛生學系暨研究所/碩士(2013年)

摘要

前言
最近研究發現，飲水砷暴露會增加上泌尿道泌尿上皮癌之危險性，但砷於體內的甲基化代謝能力是否會影響上泌尿道泌尿上皮癌之易感受性仍不清楚。p14、p15和p16是調控細胞週期進行的重要基因，過去研究指出p14、p15和p16缺失及其啟動子過甲基化與泌尿上皮癌有關，但與上泌尿道泌尿上皮癌的關係尚未瞭解。因此，本研究目的為探討砷甲基化、p14、p15和p16缺失及其啟動子過甲基化和上泌尿道泌尿上皮癌的關係。
方法
本研究為以醫院為基礎之病例對照研究，研究對象來自臺灣大學附設醫院、臺北醫學大學附設醫院及臺北市立萬芳醫院，共 213 位經組織病理學確認之上泌尿道泌尿上皮癌病例，及 213 位與病例配對年齡及性別之健康對照。在研究對象瞭解研究目的並取得同意書後以標準化問卷訪視個案，並收集血液及尿液檢體，其中 46 位病例收集腫瘤組織及鄰近正常組織。利用高效能液相層析儀串聯氫化式原子吸收光譜儀測量尿液中三價砷、五價砷、單甲基砷酸和雙甲基砷酸濃度。以聚合酶鏈鎖反應和即時甲基特異性聚合酶鏈鎖反應分析p14、p15 和p16之缺失與啟動子過甲基化。
結果
高教育程度、偶爾飲酒、喝茶及喝咖啡顯著降低上泌尿道泌尿上皮癌危險對比值，而抽菸則顯著增加上泌尿道泌尿上皮癌危險對比值。病例組比起對照組有較高的尿液總砷濃度和無機砷百分比，且在調整年齡、性別與其他基本人口學資料和生活習慣後，尿液總砷濃度高和單甲基化砷酸百分比低，顯著增加上泌尿道泌尿上皮癌之危險對比值，且呈劑量效應關係。在多變項分析中，尿液總砷濃度高同時抽菸時，顯著增加上泌尿泌尿上皮癌危險對比值；尿液總砷濃度低同時喝茶時，則有較低的危險對比值；尿液總砷濃度低、無機砷百分比低或雙甲基砷酸百分比高同時喝咖啡時，亦有較低的危險對比值。此外無機砷百分與腫瘤分級為負相關，但與膀胱復發為正相關。雙甲基砷酸百分比亦與腫瘤分級為正相關。p14啟動子過甲基化與膀胱復發和遠處轉移有關，p16過甲基化也與遠處轉移有關。在調整年齡與性別後，p16異常分別與上泌尿道泌尿上皮癌的分期和分級有邊緣性關係。
結論
本研究結果顯示尿液總砷濃度和砷甲基化代謝能力與上泌尿道泌尿上皮癌有關，也與腫瘤分級和膀胱復發有關。p16啟動子過甲基化與上泌尿道泌尿上皮癌之遠處轉移有邊緣性關係，p16異常則與腫瘤分期和分級有邊緣性關係。

關鍵字

上泌尿道泌尿上皮癌、砷甲基化、p14、p15、p16、DNA 過甲基化、基因缺失

並列摘要

Backgrounds
Recent study demonstrated that upper urinary tract urothelial carcinoma (UTUC) was associated with arsenic exposure in drinking water. But it still remains unclear whether arsenic methylation capability is related with UTUC or not. p14, p15, and p16 are important genes that regulate cell cycle. Previous studies showed that gene deletion and hypermethylation of p14, p15, and p16 promoter were related to urothelial carcinoma, but the association between alteration of p14, p15, and p16 gene and UTUC has never been proved. The aim of present study was to explore the relationships among arsenic methylation capacity, gene deletion and hypermethylation of p14, p15, and p16 promter and UTUC.
Methods
We recruited 213 pathologically proven UTUC cases and 213 age-sex matched control subjects based on a hospital case-control study from National Taiwan University Hospital, Taipei Medical University Hospital, and Taipei Municipal WanFang Hospital. Well-trained interviewers mentioned the study aim to all participants and received their formed consent, and then carried out standardized personal interview based on a structural questionnaire, and peripheral blood and urinary samples were collected simultaneously. In 46 cases, we also collected their tumor tissue and adjacent normal tissue. Concentrations of urinary arsenic species, including arsenite (As3+), arsenate (As5+), monomethylarsonic acid (MMA5+), and dimethylarsinic acid (DMA5+) were determined by a high-performance liquid chromatography-linked hydride generator and atomic absorption spectrometry (HPLC-HG-AAS). Gene deletion and promoter hypermethylation were examined by polymerase chain reaction (PCR) and real time-methylation specific polymerase chain reaction (RT-MSP), respectively.
Rssults
Study participants who had high educational levels, and alcohol, coffee or tea consumption had a significantly low odds ratio (OR) of UTUC, while those with cigarette smoking had a high OR. UTUC cases had a greater urinary total arsenic levels and inorganic arenic percentatge than healthy controls. After adjusted for confounders, we found a positive or negative dose-response relationship between total arsenic or MMA percentage and UTUC. In multivariate analysis, participants had higher urinary total arsenic and with cigarette smoking status, had a higher OR of UTUC than lower urinary total asrsenic and without cigarette smoking status. Lower urinary total arsenic and had tea or coffee comsuption had a lower OR of UTUC than higher urinary total arsenic and without tea or coffee comsuption. Arsenic methylation capability was negatively correlated with tumor grade, but had a positive correlation with UTUC recurrence. Promoter hypermethylation of p14 was associated with UTUC recurrence and metastasis. Promoter hypermethylation of p16 was also associated with metastasis. After adjumstment for age and gender, p16 abnormality had borderline relationship with tumor stage and grade of UTUC.
Conclusions
We found that urinary total arsenic levels and arsenic methylation capability were associated with UTUC, and also found that urinary total arsenic levels and arsenic methylation capability were associated with tumor grade and UTUC recurrence. Promoter hypermethylation of p14 and p16 were associated with prognosis of UTUC. Abnormality of p16 had borderline relationship with tumor stage and grade of UTUC.

並列關鍵字

Upper urinary tract urothelial carcinoma (UTUC)、arsenic methylation、p14、p15、p16、DNA hypermethylation、gene deletion

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