| Abstract: | 肺癌是世界上癌症死亡率最高的疾病約20%因癌症死亡者罹患肺癌。在台灣肺癌的死亡率位居男性癌症死亡原因第二位只少於肝癌1.4個百分點但在女性的死亡率卻高居首位綜合男性與女性的癌症死亡原因仍以肺癌為第一。抽煙與肺癌有密切的關係大約有90%的肺癌與抽煙有關。台灣的女性肺癌約佔肺癌罹患率之1/3其中90%以上之女性肺癌患者從未吸過癌。肺癌可分為四大類：小細胞肺癌大細胞肺癌鱗狀細胞肺癌和肺顯癌。抽煙與肺癌、鱗狀細胞肺癌有密切的關係與肺腺癌較無關。大約70%的台灣女性肺癌為肺腺癌，值得一提的是男性罹患肺腺癌的機率也在攀昇中。過去的肺癌研究都注重於與抽煙有關之肺癌因此對於非抽煙罹患肺腺癌的原因一直都不了解。 為了解肺腺癌致病過程必需更進一步了解與肺癌有關的細胞生物學與分子生物學。因此過去幾年我們實驗室致力於尋找在肺腺癌與正常肺組織中表現有差異的基因。為了達到這個目標我們利用抑制性的精簡雜交反應(suppressive subtractive hybridization)建立了兩個肺腺癌的精簡雜交基因庫；其中之一含有正常組織表現較高之基因(含可能的抑癌基因)另一個基因庫則含有肺腺癌表現較高的基因(含可能的致癌基因)。由於製備這些基因庫需要利用聚合脢反應(Polymerase chain reaction, PCR)因此每一個基因的數目都會被放大好幾倍。為了進一步鑑定基因庫裡的基因是否在正常組織及肺癌組織中有差異的表現量我們發展了一個差異表現量基因的篩選法。此方法不僅可以初步篩選出於此二基因庫裡有差異表現量的基因並且可以將同一個基因重複多次的clone剔除掉。用此方法我們將好幾千個clones精簡至將近三百個基因。這些clones帶有基因片段而有些片段可能來自同一個基因的不同位置。經過定序後我們可以進一步的刪掉一些來自同一個基因的clones最後我們得到113個基因。 欲進一步了解這些基因是否與肺癌的形成有關我們將這些基因和幾個已知的資料庫進行比對這些資料庫包括NCBI的EST database和SAGE database,和由周成功教授利用國外之肺癌微矩陣整理出來的資料庫http://61.218.37.60/cancer.html。分析的結果, 大約有40個基因在肺腺癌與肺正常組織中顯示出差異的表現量。 NCBI及周成功教授之資料庫來自西方國家的病人資料。我們因而想知到這40個基因在台灣女性肺腺癌病人裡是否也有特殊的表現型態。正好NHRI之黃奇英博士進行了一組非常珍貴的微矩陣分析此分析包括20組女性肺腺癌和5組男性肺腺癌病人之腫瘤及鄰近之正常組織。透過黃博士之鼎力相助我們將36個基因進行本土肺腺癌病人基因表現的分析。其中有20 個基因在本土之肺腺癌中有很顯著的差異表現量有7個基因不包括在所分析的晶片上。另外有9個基因沒有顯著的差異表現量這些可能是因為西方人和被測試之台灣人之間肺癌的差異性。那些沒有微矩陣分析資料的基因我們直接進行反轉錄-同步定量PCR反應(RT-realtime PCR)。 我們目前經過層層分析所得到的基因應該在台灣女性肺腺癌病人癌化上扮演著重要的角色。所發現的這些基因數目已遠遠超過個別實驗室所能掌握,很幸運的有些研究者對這些基因感到興趣,因而挑選一些基因進行個別研究。希望這些基因能在多個實驗的努力之下很快的開化結果。 為了研究感興趣的個別基因之功能我們想利用RNA干擾(RNA interference)技術將癌細胞株裡的感興趣之基因表現量降低再觀察這基因不表現之後對細胞的生長、存活、及轉移之影響。由於當時市面上尚未有可以用來構築穩定進行RNA干擾的載體因此我們自己利用pCDNA3.1載體發展了一個穩定度很高的RNA干擾的載體。傳統上構築RNAi模板(template)時利用兩條全長的引子(oligonucleotides,約65mer)這全長的引子攜帶著模板全長的序列。這個方法雖然十分簡單但因為需要很長的引子因此合成引子時必須進行大規模的反應然後再進行純化全長引子的步驟。大規模的合成及純化都需要較高的費用,除此之外越長的引子其合成過程很容易產生錯誤,因此當這些引子接入載體後需要進行相當多的定序反應。由於我們想利用RNAi技術進行功能性的篩選因此我們發展了一個效率高又經濟的方法取代傳統的方法進行RNAi載體的構築。這個新的RNAi載體構築方法已發表並於多個實驗室成功的使用過。利用此法可以使整個過程所需費用降低至原來之30%。 我們實驗室選了幾個基因正積極的進行基因功能的分析主要的方法是利用RNA干擾將癌細胞中感興趣的基因表現量降低或在癌細胞中過度表？
Lung cancer has been the leading cause of cancer deaths worldwide, and it accounts for about 20% of total cancer deaths. The mortality rate of lung cancer has also been the highest in Taiwan. Cigarette smoking is attributed to about 90% of lung cancer. In Taiwan, about 30% of lung cancers are female and more than 90% of them have never smoked. Among four types of lung cancers (small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma) small cell carcinoma and squamous cell carcinoma are strongly related with cigarette smoking, while adenocarcinoma and large cell carcinoma are less strongly related with cigarette smoking. About 70% of female lung cancers in Taiwan are adenocarcinoma. It is worth noting that the incidence rate of male adenocarcinoma has been increased lately. In the past, most of the lung cancer research is focused on smoking strongly related lung cancers. The mechanism of developing lung adenocarcinoma in non-smokers is still unknown. To study carcinogenesis of lung adenocarcinoma requires advance knowledge in cell biology and molecular biology of lung cancers. Thus, the past few years we focused on identifying genes that have differential expression levels between lung adenocarcinoma and its adjacent normal counterpart. To reach the goal, we established two subtractive cDNA libraries via suppressive subtractive hybridization; one library contains enriched genes preferentially expressed in lung adenocarcinoma and the other library contains enriched genes preferentially expressed in normal lung tissue. Since these subtractive cDNA libraries were established through several rounds of PCR reaction, copy numbers of each interested genes were amplified. We developed a differential screening method not only to identify differentially expressed genes but also to rule out the redundant copies of individual gene in the libraries. After sequencing analysis, the genomic information of few hundred clones was obtained through database analyses and the redundant clones were eliminated. To further identify candidate genes, we utilized the available databases (EST database and SAGE database of NCBI, and microarray database: http://61.218.37.60/cancer.html) to analyze the expression levels of each differentially expressed gene among lung cancers and normal lung. About 40 candidate genes were selected as a result of these prior analyses. These candidate genes showed significant differential expression levels between lung cancers and their adjacent normal lung tissues from western lung cancer patients. We do not know whether the expression patterns of these genes are also significant among female lung adenocarcinomas in Taiwan. Dr. Chi-Yin Huang (NHRI) performed valuable microarray analyses on a set of 20- female lung adenocarcinomas and 5-male lung adenocarcinomas. With Dr. Huangs generous assistance, these candidate genes were further analyzed in their microarray database. Among 36 genes analyzed in the NHRI microarray, 20 of them showed significant differential expression patterns between lung cancers and normal lung tissues and 7 genes were not on the analyzed chip. The rest of clones selected from western database did not show significant differential expression patterns among Taiwanese female lung cancers tested. Genes that were not analyzed in this set of chip were directly analyzed by RT-realtime PCR. The number of identified candidate genes was far from we can handle in the individual laboratory. Fortunately, several researchers were interested in some of the genes and selected for further studies. Hopefully this study would have fruitful results in the near future. To study the biological function of these candidate genes, we used RNA interference to knock d |
