臺灣博碩士論文加值系統

壹、中文摘要
肺癌是台灣地區男性及女性癌症死亡原因的首位，但流行病學資料顯示國人吸煙的人口比例卻顯著與西方國家不同，因此先天遺傳敏感性與香菸以外的環境因素，可能是造成台灣地區肺癌形成的原因。由於本研究室在先前的實驗中證實，台灣肺癌形成的過程中常伴隨著基因不穩定的現象。利用微衛星不穩定（microsatellite instability, MI）即可偵測到基因不穩定的現象，而造成MI的原因有可能是DNA進行複製時聚合酵素發生問題；或者是DNA在複製錯誤後，錯誤配對修補基因 (mismatch repair gene) 功能上有問題，無法將有錯誤的鹼基配對修補，因此造成一連串功能基因的異常。所以本研究分析了兩個主要的配對修補基因hMLH1和hMSH2，分別對其蛋白質表達的情形、mRNA表達的情形、hMLH1啟動子甲基化的情形、還有異質性缺失 (loss of heterozygosity；LOH) 做偵測，並探討其形成的可能機制，另外也一併進行與病理資料、其他分子證據如微衛星不穩定性 (MI) 做相關性的探討。
蛋白質方面，採用免疫組織染色法（immunohistochemistry）針對錯誤修補系統（mismatch repair system, MMR）之hMLH1、hMSH2 蛋白表現進行分析，發現有高達56.8％ (96/196) 的病人其hMLH1 蛋白有不表達的情形、而hMSH2蛋白僅有17.1％ (13/76) 不表達。mRNA方面，以RT-PCR分析其mRNA的表達情形，發現在hMLH1 mRNA方面亦有55.8% (43/77) 的高比例病人不表達、hMSH2 mRNA則為15.6% (12/77) 不表達；hMLH1 和hMSH2 其mRNA與蛋白質表達有相當高的一致性 (hMLH1 , P=0.001；hMSH2 , P=0.001)。
本實驗進一步探討，蛋白質的不表現、mRNA的不表達的可能原因。在DNA層次方面，若在基因啟動子序列部分有高度甲基化的情形，常會導致轉錄因子和RNA 聚合酵素無法結合到啟動子的位置，轉錄 (Transcription) 就無法進行，mRNA就無法合成。因此，本研究進一步的分析hMLH1蛋白、mRNA不表達是否是因為hMLH1的啟動子過度甲基化所致。結果顯示hMLH1啟動子高度甲基化的病人中，其hMLH1蛋白沒有表達的比例高達73.8% (45/61)；hMLH1啟動子高度甲基化的病人中，hMLH1 mRNA 不表達的比例82.4% (28/34)。顯示了，hMLH1蛋白不表達的原因可能是其啟動子高度甲基化進而導致mRNA不表達所致 (P=0.001)，也顯示了在台灣地區非小細胞肺癌的形成機制中，錯誤配對修補基因hMLH1啟動子高度甲基化，扮演了的角色是不容忽視的。另外，本實驗也使用基因座缺失（loss of heterozygosity, LOH）的分析方法，針對hMLH1所在的染色體區位3p21的微衛星序列 D3S1768，進行LOH的分析，發現其中有高達56.4% (22/39) 的病人其hMLH1所在的染色體區位有LOH的情形產生；研究結果亦指出，在hMLH1基因的位置，同時有LOH和啟動子高度甲基化的病人，有50％ (8/16) 的比例，顯示了LOH和啟動子高度甲基化，對hMLH1蛋白質表達異常有一定的影響力。
接著，進一步偵測hMLH1蛋白表現和抑癌基因 (tumor suppressor genes) 的不表現如p16蛋白、pRb蛋白等和p53基因的突變情形的相關性，以探討hMLH1扮演致變子 (mutator) 的可能性；初步發現，hMLH1蛋白變異與其他檢測的基因變異並無明顯相關。在hMLH1基因或蛋白質變異與微衛星不穩定性 (MI) 的分析方面，在微衛星不穩定的病人中其hMLH1蛋白不表達的比例高達73% (27/37)。顯示hMLH1蛋白質不表達，很可能是導致微衛星不穩定的主要原因之一 (P=0.025)；另外，微衛星不穩定性的病人也可能和異質性缺失有一定程度的相關性 (P=0.092)，至於其他分析資料，可惜因人數過少，未達統計上的意義。
由本研究結果可推測：錯誤配對修補基因hMLH1之變異可能參與台灣地區肺癌之形成，並導致微衛星不穩定性；hMLH1變異機制主要是因為啟動子高度甲基化導致mRNA不表達，進一步致使蛋白質也不表達；另外，異質性缺失 (LOH) 可能也是另一變異機制。並由hMLH1、hMSH2兩錯誤配對修補基因的異常表達情形比較可知，可能hMSH2在台灣肺癌形成機制中僅佔一較次要的角色。而其他本研究未探討的錯誤配對修補基因啟動子高度甲基化和異質性缺失現象研究，在未來皆是很值得深入探討的主題。

貳、英文摘要
Lung cancer is the leading cause of cancer deaths in Taiwan. Genetically determined variation in DNA repair capacity is thought to contribute to susceptibility to tobacco-related cancers, such as lung cancer. In addition, defects in mismatch repair (MMR) genes have been implied in several types of sporadic and hereditary cancers in terms of inducing microsatellite instability (MI) of tumor cells. We have previously found that MI occurs in 41% of non-small cell lung cancer patients (NSCLC). However, there was only little report discussing the MMR gene alteration in lung cancer.
In order to elucidate the role of MMR in human lung carcinogenesis in Taiwan, we examined the alteration of two DNA mismatch repair genes, hMLH1 and hMSH2, in primary lung tumor tissues from NSCLC patients. We investigated the expression levels of hMLH1 and hMSH2 proteins in 169 and 77 NSCLC tumors , respectively, by the immunohistochemical analysis. Ninety-six (56.8%) patients had the alteration of hMLH1 protein expression; thirteen patients (17.1%) had the alteration of hMSH2 protein expression. In addtion, we found that the alteration of protein expression correlated with the aberrant mRNA expression by RT-PCR assay for both hMLH1 and hMSH2 (P=0.001). The alteration frequencies of mRNA expression were 55.8% and 15.6% for hMLH1 gene and hMSH2 gene, respectively.
To further examine the role of promoter hypermethylation in the identified alteration of protein expression, we also examine the association between hMLH1 promoter hypermathylation and hMLH1 protein expression. Forty-five (73.8%) patients containing hMLH1 promoter hypermethylation showed the alteration of protein expression (P = 0.001). The results suggested that mismatch repair plays a significant role in NSCLC tumorigenesis in Taiwan and that hMLH1 promoter hypermethylation is closely relative to the altered expression of mRNA and protein in the hMLH1 mismatch repair gene. Furthermore, we conducted a loss of heterozygosity (LOH) analysis at 3p21 microsatellite polymorphic marker D3S1768 for the deletion of hMLH1 region in a series of 39 NSCLC patients. The high percentage of LOH (56.4%) was observed at the D3S1768. The result suggested that loss of chromosome 3p21 may be also a critical event in the pathogenesis of lung cancer in Taiwan.
To investigate the association of hMLH1 alteration with MI, we also studied the correlation between MI and expression of hMLH1 mismatch repair protein in 73 patients. Twenty-seven of 37 MI-positive patients (73.0%) did not express hMLH1 protein. The data showed that MI was associated with altered hMLH1 expression (P=0.025). In addition, there was a tendency of association between LOH at D3S1768 (3p21) region and MI (P=0.092). The correlation suggests the alteration of the hMLH1 gene/ protein may be associated with microsatellite instability (MI) because of the failure to correct replication slippage errors.
In conclusion, our data suggest that alteration of hMLH1 gene and/or protein involved in lung tumorigenesis in Taiwan. The major alteration mechanism may be the promoter methylation of hMLH1 gene, and therefore inactivating its mRNA and protein expression. Moreover, LOH of the hMLH1 gene can also play an important role in alteration of hMLH1 gene. As regards hMSH2 gene, the data indicated that hMSH2 does not play a major etiological role in lung tumorigenesis in Taiwan. Validating the promoter hypermethyaltion and LOH in other mismatch rapair genes are worthy of further investigation in lung cancer tumorigenesis in Taiwan.

目 錄
錯誤配對修補基因變異
參與台灣地區肺癌形成之機制
Genetic Alterations of Mismatch Repair Genes
in Lung Tumorigenesis in Taiwan
壹、中文摘要 -------------------------------------------------------1
貳、英文摘要 -------------------------------------------------------4
參、文獻總論 -------------------------------------------------------6
一、引言 -------------------------------------------------------6
二、肺癌的分類及檢體種類--------------------------------9
三、基因不穩定與肺癌之關係---------------------------------9
四、錯誤配對修補機制與癌症形成的關係---------------12
五、何謂啟動子高度甲基化 (Promoter
Hypermethylation) 及其影響---------------------------16
六、何謂異質性缺失
(loss of heterozygosity, LOH)--------------------------18
肆、研究目標 -----------------------------------------------------21
一、偵測並探討錯誤配對修補基因hMLH1、hMSH2其蛋白質表達情形參與台灣地區非小細胞肺癌 (NSCLC) 形成之機制-------------------------------21
二、偵測並探討錯誤配對修補基因hMLH1、hMSH2其mRNA表達情形參與台灣地區非小細胞肺癌 (NSCLC) 形成機制的情形-------------------------21
三、研究台灣地區非小細胞肺癌病人中錯誤配對修補基因hMLH1其啟動子高度甲基化(Promoter Hypermethylation)的情形---------------------------22
四、探討錯誤配對修補基因hMLH1表達與否與異質性缺失 (LOH) 的情------------------------------------23
五、探討hMLH1蛋白質表達程度、mRNA 表達情形、啟動子高度甲基化狀態 (Promoter Hypermethylation) 異質性缺失 (LOH) 與微衛星序列不穩定 (MI) 間在參與台灣地區非小細
胞肺癌 (NSCLC) 形成機制的相關性--------------23
伍、方法總論 ------------------------------------------------------25
一、檢體來源及病例資料 --------------------------------25
(1)、本研究檢體來源 ---------------------------------25
(2)、LOH分析用檢體來源 ---------------------------26
二、外科切除組織塊之保存 -------------------------------27
(1)、液態氮保存 ----------------------------------------------27
(2)、.臘塊保存 -------------------------------------------------27
三、DNA之製備 ----------------------------------------------28
(1)、啟動子高度甲基化 (Promoter methylation)、
微衛星不穩定性 (MI) 分析用-----------------28
(2)、LOH分析用 --------------------------------------28
四、hMLH1 & hMSH2蛋白表現之分析-----------------30
(1)、免疫組織化學染色分析
( Immunohistochemistry , IHC) ------------------30
(2)、染色切片的判讀標準 --------------------------31
五、RNA & cDNA之製備----------------------------------32
六、hMLH1 & hMSH2 RNA 表現程度之分析---------32
(1)、聚合鍊連鎖反應 ( Polymerase chain reaction, PCR ) ------------------------------------------------33
(2)、mRNA表現之分析--------------------------------34
七、hMLH1啟動子甲基化 (Promoter methylation)
程度分析-------------------------------------------------35
八、LOH分析-------------------------------------------------37
九、MI分析 --------------------------------------------------38
十、統計分析 ------------------------------------------------39
陸、結 果 ---------------------------------------------------------41
一、探討台灣地區非小細胞肺癌病人(NSCLC)其hMLH1蛋白的表現情形與mRNA表現程度、啟動子高度甲基化 (promoter hypermethylation)、異質性缺失(LOH)、肺癌病人病理分類、預後及微衛星不穩定性(MI)、、其他抑癌基因變異之相關性 -------------41
(一)、探討台灣地區非小細胞肺癌病人其hMLH1蛋白的表現情形---------------------------------41
(二)、探討台灣地區非小細胞肺癌病人其hMLH1的蛋白異常表現與病理資料的相關性---------42
(三)、探討台灣地區非小細胞肺癌病人其hMLH1的蛋白表達情形和mRNA表達情形的相關性 ------------------------------------------------------43
(四)、探討台灣地區非小細胞肺癌病人其hMLH1的蛋白異常表現與其啟動子高度甲基化的相關性 ---------------------------------------------------43
(五). 探討台灣地區非小細胞肺癌病人其hMLH1的蛋白異常表現與其異質性缺失(LOH)
的相關性 ------------------------------------------43
(六)、探討台灣地區非小細胞肺癌病人其hMLH1蛋白異常表現與微衛星不穩定的關係性 ------44
(七)、探討台灣地區非小細胞肺癌病人其hMLH1
的蛋白異常表現與其他抑癌基因 (p53、p16 and pRb) 異常的相關性 ------------------------44
(八). 探討台灣地區非小細胞肺癌病人其hMLH1的蛋白異常表現與預後的相關性 ---------------45
二、探討台灣地區非小細胞肺癌病人(NSCLC)其hMLH1 mRNA的表現情形與啟動子高度甲基化程度、肺癌病人病理分類之相關性--------------------------------46
(一)、探討台灣地區非小細胞肺癌病人其hMLH1 mRNA的表達情形 -----------------------------46
(二)、探討台灣地區非小細胞肺癌病人其hMLH1的mRNA異常表現與病理資料的相關性--------46
(三)、探討台灣地區非小細胞肺癌病人其
hMLH1 mRNA不表現與其蛋白質不表現
及啟動子高度甲基化程度的相關性---------47
三. 探討台灣地區非小細胞肺癌病人(NSCLC)其hMLH1啟動子高度甲基化程度與肺癌病人病理分類、微衛
星不穩定性(MI)、和異質性缺失(LOH)之相
關性 ---------------------------------------------------------48
(一)、探討台灣地區非小細胞肺癌病人其hMLH1
啟動子高度甲基化的程度比例-----------------48
(二)、探討台灣地區非小細胞肺癌病人其hMLH1
啟動子高度甲基化程度與病理資料的相關性--------------------------------------------------------48
(三)、探討台灣地區非小細胞肺癌病人其hMLH1
啟動子高度甲基化與其異質性缺失 (LOH) 的相關性 ---------------------------------------------49
(四)、探討台灣地區非小細胞肺癌病人其hMLH1
啟動子高度甲基化與微衛星不穩定的關係性 --------------------------------------------------------50
四、探討台灣地區非小細胞肺癌病人(NSCLC)其hMLH1 LOH情形與肺癌病人病理分類、微衛星不穩定性之相關性 ----------------------------------------------------51
(一)、探討台灣地區非小細胞肺癌病人其hMLH1 LOH的情形 -------------------------------------51
(二)、探討台灣地區非小細胞肺癌病人其hMLH1 LOH異常與病理資料的相關性 -------------52
(三)、探討台灣地區非小細胞肺癌病人其hMLH1 LOH與微衛星不穩定的關係性 ---------------52
五、探討台灣地區非小細胞肺癌病人(NSCLC)其
hMSH2蛋白的表現情形與mRNA表現程度、
肺癌病人病理分類之相關性 -------------------------53
(一)、探討台灣地區非小細胞肺癌病人其hMSH2蛋白的表現情形--------------------------------------53
(二)、探討台灣地區非小細胞肺癌病人其hMSH2的蛋白異常表現與病理資料的相關性-----------53
(三)、探討台灣地區非小細胞肺癌病人其hMSH2
的蛋白表達情形和mRNA表達情形的一致性 --------------------------------------------------------54
六、探討台灣地區非小細胞肺癌病人(NSCLC)其hMSH2 mRNA的表現情形與肺癌病人病理分類之相關性 --------------------------------------------------------------54
(一)、探討台灣地區非小細胞肺癌病人其hMSH2 mRNA的表達情形------------------------------54
(二)、探討台灣地區非小細胞肺癌病人其hMSH2的mRNA異常表現與病理資料的相關性 ------------------------------------------------------55
柒、討 論 -----------------------------------------------------------56
一、探討hMLH1 基因/蛋白變異參與台灣地區非小細胞肺癌（NSCLC）的形成與其機制----------------56
二、探討hMLH2 基因/蛋白變異參與台灣地區非小細胞肺癌（NSCLC）的形成及其機制-----------------63
三、研究應用與未來工作 ----------------------------------65
捌、參考文獻 -----------------------------------------------------66
玖、圖 表 -----------------------------------------------------------75
圖 目 錄
圖一 Figure. 1 Representative figures of immunohistochemical analysis of hMLH1 protein ----------------------------75
圖二 Figure 2. Representative figures of immunohistochemical analysis of hMSH2 protein ----------------------------76
圖三 Figure 3. The Kaplan-Meier survival curves with respect to hMLH1 protein expression.---------------------------77
圖四 Figure 4. Representative figures of hMLH1 and hMSH2 mRNA expression analysis.-------------------------------------78
圖五 Figure 5. Representative figures of promoter hypermethylation analysis of hMLH1 gene in normal lung tissue------------------------------------------------------79
圖六 Figure 6. Representative figures of promoter hypermethylation analysis of hMLH1 gene in tumor lung tissue------------------------------------------------------80
圖七Figure 7. Representative figures of LOH analysis of chromosome 3p21 using the D3S1768 microsatellite sequence ------------------------------------------------------------81
表 目 錄
表一 Table 1 Clinical features of hMLH1 protein expression
-positive and-negative patients--------------------82
表二 Table 2 hMLH1 protein expression in relation to genetic alterative factors------------------------------------------83
表三 Table 3 Clinical features of hMLH1 mRNA expression
-positive and-negative patients -----------------------84
表四 Table 4 Clinical features of hMLH1 promoter methylation -positive and-negative patients------------------------85
表五 Table 5 Clinical features of hMLH1 LOH -positive and-negative patients---------------------------------------------------86
表六Table 6 Clinical features of hMSH2 protein expression
—positive and-negative patients-----------------------87
表七Table 7 Clinical features of hMSH2 mRNA expression
-positive and-negative patients ------------------------88
表八Table 8 The correlation of hMLH1 protein expression , promoter hypermethylation and mRNA expression -------------------------------------------------------------89

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