2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/37919The goal of this project is to identify putative oncogenes and tumor suppressor genes that are responsible for lung carcinogenesis. Recently, the incidence rate of lung cancer increases significantly. Similar to the rest of the world, the mortality rate of lung cancer is the highest among cancer death 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 major types of lung cancers (small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and adenocarcinoma), adenocarcinoma and large cell carcinoma are less strongly related with cigarette smoking. However, 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. Thus, study genes expressed differentially between lung adenocarcinoma and normal lung tissue would increase our understanding in carcinogenesis of lung adenocarcinoma, and hopefully would provide new information for development of early diagnosis techniques, and better therapeutic strategies. Two subtracted cDNA libraries were established via subtracted cDNA hybridization between female lung adenocarcinoma and its normal counterpart lung tissue; N-T subtracted cDNA library contains enriched putative tumor suppressor genes, and T-N subtracted cDNA library contains enriched putative oncogenes. After differentially screenings and sequencing, 76 and 37 independent cDNA clones are selected from N-T and T-N subtracted cDNA libraries, respectively. The differential expression patterns of these genes between lung cancer and normal tissues in lung cancer patients were confirmed by following two techniques. First, perform realtime RT-PCR reaction of candidate genes in pair of lung cancer and normal tissues from various patients. Second, amplify each cDNA clone and generate cDNA microarray for screening by using cDNA probes generated from patients？H？H tissues. Realtime RT-PCR can accurately reflect the differential expression levels of the target genes between normal and cancer tissues. However, the cost of primers and reagent and time consuming are the major disadvantages of this technique. To compensate realtime RT-PCR, we generated the subtracted cDNA microarray for patient screenings. At this moment, the signals hybridized with digoxgenin labeled probes are not strong enough to differentiated each cDNA clones. Thus, we plane to amplify these probes with gene specific primers by linear PCR amplification. We have selected six genes for further studies. Full-length clones are under construction. Furthermore, we generated a vector for generating stable RNAi molecules in human cells. Thus, we plane to study the effect of over-expression or knockout of target genes on cell growth, death and on ability of tumor formation. In addition, antibodies of the candidate proteins will be generated for further biochemical studies and their sub-cellular localization studies.本計劃之主要目標是探討肺腺癌病人的肺正常組織與癌組織之間基因表現的差異性。近數十年來，全世界罹患肺癌的病人顯著的增加。在台灣，肺癌的死亡率位居於男性癌症死亡原因第二位，僅少於肝癌1.4個百分比，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中仍以肺癌為第一。抽煙與肺癌有密切的關係。鱗狀細胞癌與小細胞癌和抽煙有密切的關係，與肺腺癌較無關。而絕大多數的台灣女性肺癌病人中以肺腺癌的罹患率最高，這些人絕大多數不抽煙。值得注意的是近年來男性肺腺癌病人的發生率也逐漸的居於領先的局勢。因此，探討肺腺癌於正常肺組之間基因表現的差異性，將有助於我們對肺腺癌形成的了解，並期望對於肺腺癌的治療、診斷、或預防上提供一些重要的訊息。 本實驗室由肺腺癌的精簡雜交基因庫裡篩選了一些基因，這些基因可能與肺癌的形成有關。本年度，我們將針對這些各別的基因，進行較深入的研究。首先，我們將利用同步定量分析法，將這些基因於病人之正常組織與腫瘤組織進行定量分析。對於表現量有明顯差異的基因，我們將針對這些蛋白質製造抗體，以利於蛋白質的分析。另一個重要的實驗是利用RNA干擾的技術，於細胞內將這些基因的表現破壞。RNA干擾技術可以測試這些基因是否對細胞的生長速度、凋亡、甚至於致腫瘤的能力的影響。另一方面，我們將這些基因表現於表現量很低的細胞內，用以觀察當這些基因的表現量恢復時對細胞的生理現象是否有影響。肺腺癌精簡雜交基因庫抑癌基因致癌基因同步定量PCRRNA干擾Lung adenocarcinomaSubtracted cDNA librarytumorsuppressor genesoncogenesRealtime PCRRNA interferenceProject