從分析基因異常到其致病新機制的觀點來探討以消化系統癌症分子基礎的致病機轉

Molecular Basis Of Digestive Tract Cancer From Genome Aberration Analysis To Exploring A Novel Mechanism

張由俐(Yu-Li Chang)

指導教授 ： 劉大智 ； 張建國

高雄醫學大學/醫學院/醫學研究所博士班/博士(2016年)

摘要

消化系癌症通常是一種遺傳性或後天基因表現變異導致異常性細胞生長的疾病，常以侵入或擴散方式到身體的其他部位。根據WHO 2015年公告預計未來20年五大癌症病例數會再上升70％。消化系癌症在這五大癌症中就佔了二位。然致病原因與機轉至今仍是相當重要的探討方向。因此，我們嘗試由非編碼核糖核酸分子及基因量表現及突變點改變這幾項因子來探討對於消化系癌症發生及進展的相關性。本論文分二階段主題進行。第一階段研究主題分兩部分:即食道癌的染色體分析，與利用HRM的方法分析口腔癌及大腸癌相關基因突變。第二階段著重於X染色體上XIST中非編碼核糖核酸小分子新的機轉探討。

第一階段研究主題第一部分
首先我們聚焦在消化系癌症中的食道癌(esophageal cancer; EC)並作一系列全基因篩檢。針對全部染色體做全面性比對。目的在於檢測細胞遺傳學改變在食道癌（EC）的基因熱點區(異常頻率高)。共收集了40例原發性EC及其附近配對非腫瘤組織。利用全基因組雜交比較法（slide comparative genomic hybridization; CGH）確認染色體片段的損失或增加。在癌症周邊組織中發現，染色體異常增加片段在19p（5/40，13％），20q（5/40，13％），並在9p（13/40，33％），12q（10/40，25％），13q（10/40，25％），5q（9/40，23％），6q（9/40，23％），7q（9/40，23％）和8p（9/40，23％）這些染色體異常處找到異常基因缺失。在癌症組織中發現，染色體異常增加片段在8q (10/40, 25%), 3q (9/40, 23%), 2q (7/40, 18%),及13q (7/40, 18%),染色體異常損失片段是在1q（8/40，20％），4q（8/40，20％），3p（7/40，18％），5q（7/40，18％），和18q（7/40，18％）的位置。統計比較之後，發現兩種組織的增加及缺失同時的熱點區是在1q, 2p, 3p, 5q, 6q, 9p, 11p, 15q, 16q, 18q, 21q,及20p。非癌症缺失的熱點區是在-4p, -7q, -8p, -10q, -12q, -13q, 和-14q 。癌症缺失的熱點區是在 +17p, +19q, +22q, +1q, +2pq, +3q, -4q, +4q, +5q, 7p, +8q, +10q, +12q, +13q, +14q -17p, -19pq, 和-22q 。從這些結果，我們認為最鄰近EC的正常部位的組織雖經病理鑑定正常仍出現染色體異常應可以被認為是一個癌前變化區域。因為癌症和非癌症組織之間的變化可能是在基因異常下造成染色體改變。非癌症組織出現的異常熱點有一部分是目前文獻不曾報告過，因此我們認為這或許是我們自己族群會出現的染色體異常熱點區。

第一階段第二部分
由第一階段研究主題結果得知消化系癌症中台灣族群口腔食道癌症基因在染色體有異常高頻區。經由統計後並挑取染色體高異常熱區其中一個基因-TNFAIP3基因，因為TNFAIP3基因是NF-kB途徑中極重要的負調節物，並且在淋巴瘤和自身免疫疾病經常造成失活或突變甚至缺失。我們檢測TNFAIP3在口腔鱗狀細胞癌（oral squamous cell carcinoma; OSCC）失活的頻率。 由81例口腔鱗癌和50正常組織對照組比對。並利用高分辨率熔化分析法（high-resolution melting; HRM）應用於TNFAIP3突變，並將結果經DNA定序來確認。三種突變和三個單核苷酸多態性（single-nucleotide polymorphisms; SNPs）發現與OSCC有關聯性;在口腔鱗狀細胞癌病例TNFAIP3突變率約發現3.7％（八十一分之三）。除了舊有突變區外，我們發現並確定E361K被為一個新的突變以及SNP, p.P714S 不同於過去提及的點突變 (p.P714A) (rs369155845)。此外還確定了50名正常台灣人含有5個SNP，其中兩個沒有在我們的口腔鱗狀細胞癌組織中找到。我們的結果證明，TNFAIP3這個基因在我們收集台灣族群口腔鱗狀細胞癌罹患者中發生突變率極低。
接下來我們聚焦在消化系癌症中的結直腸癌(CRC)並探討在台灣人口與結直腸癌（CRC）相關的驅動程序基因突變。在這項研究中，收集了103 CRC患者進行了評估。患者有66個男性和37名女性，年齡範圍26-86歲。我們使用高分辨率熔解分析法（HRM）和直接DNA定序的CRC相關的通路的13驅動程序基因突變的特徵。在103例患者中，在13個驅動基因之約73.79％的突變。我們在APC，MLH1，MSH2，PMS2，SMAD4和TP53中發現以前文獻沒有報告發現的18個新的突變點。此外，我們比對了dbSNP癌症組織數據資料庫從APC，BMPR1A，MLH1，MSH2，MSH6，MUTYH和PMS2基因中發現有16個突變點;然而，這些突變不能在周邊血細胞中檢測到。只有APC突變發現伴有淋巴結轉移及癌症分期轉移。因此我們認為其他基因突變與臨床病理特徵之間沒有直接關聯。此外，具有兩個或多個驅動程序基因突變發現與淋巴結轉移相關。我們的研究結果證實CRC 13個驅動基因的通路驅動基因在台灣CRC的患者發展有一定的重要性。

第二階段研究主題
由第一階段研究兩個部份結果及文獻得知基因表現量及突變與消化系癌症息息相關。近期文獻更提及因小的核糖核酸分子干擾而改變基因表現量。因此我們此階段經由生物資訊比對出X染色體上一段小的核糖核酸分子。雌性哺乳動物尤其人類有兩條染色體，X染色體上在有一個非活化中心負責一個X染色體非活化作用的過程，因而兩個X由染色體中的其中之一會被非活化。整個過程主要是由一個很大的非編碼核糖核酸分子(noncoding RNA-XIST)所調控。在本研究中，我們在XIST中發現了一個新的RNA(XPi2)，它的表現及功能與X染色體非活化作用無相關性。本研究所提出的結果證明XPi2與Argonaut 及Nucleolin有交互作用及在Quadruplex的結構中都扮演重要的角色。X染色體上一段不活動特異性轉錄物（XIST）有稱一個長的非編碼RNA，是對第X染色體失活的啟動是不可少的。然而，知之甚少XIST在生理過程中真核細胞的其他角色。在這項研究中，生物信息學方法顯示，XIST可以和PIWI相互作用小的核糖核酸分子(small RNA)。我們命名此段小核糖核酸分子為XPi2。該XPi2 small RNA進行北方雜交點墨法實驗證實存在，其表達與性別表現無關，這表明此段小的核糖核酸分子的作用是超越的X染色體失活。再來結合LC-MS-MS確定了兩個此段小的核糖核酸分子相關蛋白，亦即Argonaut 及Nucleolin，這是與Quadruplex的形成相關之重要蛋白。此外，微陣列數據顯示XPi2的敲擊後會下調KRAS通路功能。我們並測試了包括KRAS基因在內的10個與Quadruplex架構相關的基因。發現我們敲擊XPi2而失去活性後這10個基因中，有某些基因的轉錄水平顯著降低。因此我們提出，XPi2可以通過改變KRAS基因Quadruplex架構來調節基因的表達。並經由Piwi 集核內蛋白與 RNA XPi2相互作用來共同調節Quadruplex的構造。我們進而收集31位大腸癌臨床病例證實XPi2與造成消化系癌症中的大腸癌之KRAS基因有致病相關性。

關鍵字

消化系統癌症 ； 分析基因異常 ； 非編碼核糖核酸小分子

並列摘要

Digestive cancer is a disease which is usually genomic or somaitc anomalies leading to uncontrollable abnormal cell growth, often invasive or diffused into other parts of the body. According to WHO, the 2015 bulletin released worldwide in cancer incidence and mortality of the main reasons for the investigation. In the next 20 years, the number of new cases will rise 70% more than the current number. Digestive cancer is among the most common forms of cancer, and two types of digestive cancer are considered to be the most deadly in Taiwan. Its association with the mechanisms of cancer development and disease progression is still a very important direction to explore. Here we try to find how the non-coding RNA molecules, gene mutation, and expression change are related to occurrence and progression of digestive cancer. There are two main topics in this thesis. The first part of the research topic is divided into two sections, i.e. (1) the esophagus chromosome analysis and utilization of the HRM method for analyzing oral cancer and colorectal cancer related gene mutations and (2) the focus on the X chromosome XIST related small RNA molecule for exploring a novel mechanism.
In section one of the first part, we focused on the detection of cytogenetic alterations in esophageal cancer (EC) which demonstrated high anomalous frequency. A total of 40 cases of primary EC and their paired nearby nontumor tissues were collected. The comparative genomic hybridization (CGH) is the technique that brings out the gains and losses of chromosome fragments and was applied to determine the aberrations from the tissue DNA. In noncancer tissues, the gains were at 19p (5/40, 13%), 20q (5/40, 13%), and losses at 9p (13/40, 33%), 2q (10/40, 25%), 12q (10/40, 25%), 13q (10/40, 25%), 5q (9/40, 23%), 6q (9/40, 23%), 7q (9/40, 23%), and 8p (9/40, 23%). In the 40 cases of primary EC, the gains were at 8q (10/40, 25%), 3q (9/40, 23%), 2q (7/40, 18%), and 13q (7/40, 18%), and the losses were at 1q (8/40, 20%), 4q (8/40, 20%), 3p (7/40, 18%), 5q (7/40, 18%), and 18q (7/40, 18%) in comparison with paired nearby noncancerous tissues. We found that the loss aberrations were on 1q, 2p, 3p, 5q, 6q, 9p, 11p, 15q, 16q, 18q, 21q and gains on 20p in both tumor and nontumor tissues; nevertheless, -4p, -7q, -8p, -10q, -12q, -13q, -14q and +17p, +19q, +22q were only found in nontumor tissues and +1q, +2pq, +3q, -4q, +4q, +5q, 7p, +8q, +10q, +12q, +13q, +14q -17p, -19pq, -22q in EC. From these results, we suggest that most of the tissues near the cancer parts of EC may be considered as a precancerous region. Changes in cancer and non-cancer tissues may be caused by abnormal chromosomal changes in genes. The appearance of some of anomalous hot spots in non-cancer tissue has not been reported in medical literature so far; therefore, we believe that the alterations between cancer and non-cancer tissues may play a role in the development of EC in the Taiwanese population.
In section two of the first part, we know that there are anomalous frequency regions in the chromosomes of the genes in oral squamous cell carcinoma (OSCC) in the Taiwanese population, and that TNFAIP3 acts as a negative regulator of the NF-?羠 pathway, and in lymphoma and autoimmune diseases, it is frequently inactivated by mutations and/or deletions. We investigated the prevalence of inactivation of TNFAIP3 in oral squamous cell carcinoma (OSCC). DNA was extracted from 81 cases of OSCC and 50 peripheral blood samples from normal controls. A high-resolution melting (HRM) analysis was used to characterize TNFAIP3 mutations, and the results were confirmed by direct DNA sequencing. Three mutations and three single-nucleotide polymorphisms (SNPs) were found to be associated with OSCC; the TNFAIP3 mutation occurred in 3.7% (3/81) of OSCC cases examined. p.E361K was identified as a novel mutation. A novel SNP, p.P714S differed from one reported previously (p.P714A) (rs369155845) at that site. We also identified five SNPs in 50 normal Taiwanese individuals, and two of them [c.296-15C>T (rs377482653) and c.305A>G (p.N102S) (rs146534657)] were not found in our OSCC tissue. HRM facilitated screening of genetic changes. In addition, our results indicate that the prevalence of the TNFAIP3 mutation is low in OSCC.

Then, we investigated the driver gene mutations associated with colorectal cancer (CRC) in the Taiwanese population. In this study, 103 patients with CRC were evaluated. The samples consisted of 66 men and 37 women with a median age of 59 years and an age range of 26-86 years. We used high-resolution melting analysis (HRM) and direct DNA sequencing to characterize the mutations in 13 driver genes of CRC-related pathways. The HRM assays were conducted using the LightCycleⓇ 480 Instrument provided with the software LightCyclerⓇ480 Gene Scanning Software Version 1.5. We also compared the clinicopathological data of CRC patients with the driver gene mutation status. Of the 103 patients evaluated, 73.79% had mutations in one of the 13 driver genes. We discovered 18 novel mutations in APC, MLH1, MSH2, PMS2, SMAD4 and TP53 that have not been previously reported. Additionally, we found 16 de novo mutations in APC, BMPR1A, MLH1, MSH2, MSH6, MUTYH and PMS2 in cancerous tissues previously reported in the dbSNP database; however, these mutations could not be detected in peripheral blood cells. The APC mutation correlates with lymph node metastasis (34.69% vs 12.96%, P=0.009) and cancer stage (34.78% vs 14.04%, P=0.013). No association was observed between other driver gene mutations and clinicopathological features. Furthermore, having two or more driver gene mutations correlates with the degree of lymph node metastasis (42.86% vs 24.07%, P=0.043). Our findings confirm that there is a certain degree of importance regarding the 13 CRC-related pathway driver genes in the development of CRC in Taiwanese patients.
The second part involves the following points : X-inactive-specific transcript (XIST), a long non-coding RNA, is essential for the initiation of X-chromosome inactivation. However, little is known about other roles of XIST in the physiological process in eukaryotic cells. In this study, the bioinformatics approaches revealed that XIST could be processed into a PIWI-interacting small RNA XPi2. The XPi2 RNA was confirmed by Northern blot assay, and its expression was gender-independent, suggesting that the role of XPi2 was beyond X-chromosome inactivation. The pull-down assay combined LC-MS-MS identified two XPi2-associated proteins, nucleolin and hnRNP A1, which were related to the formation of G-quadruplex. Moreover, the microarray data showed that the knockdown of XPi2 down-regulated the KRAS pathway. Consistently, we tested the expression of ten genes including KRAS, which were regulated by G-quadruplex formation and found that the knockdown of XPi2 caused a dramatic decrease in the transcription level of KRAS among the ten genes. The results of CD/NMR assay also supported the interaction of XPi2 and the polypurine-polypyrimidine element of KRAS. Accordingly, XPi2 may modulate the KRAS expression by attenuating G-quadruplex formation. Our present work sheds light on the novel role of Piwi-interacting RNA XPi2 in modulating the G-quadruplex formation which may play some roles in the KRAS related carcinogenesis. Based on the aforementioned results, we collected and studied 31 (62n) clinical cases of colorectal cancer and confirmed that there was correlation between XPi2 and KRAS, which led to the development of colorectal cancer, one of digestive cancers.

並列關鍵字

Digestive Tract Cancer ； Genome Aberration Analysis ； non-coding RNA

參考文獻

第一章

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