| 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,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)。我們發展了一個差異表現量基因的篩選法,將好幾千個clones,精簡至將近三百個基因,經過定序後得到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。 為了解這些基因在在台灣肺腺癌表現的情形,我們與NHRI之黃奇英博士合作,利用他們的微矩陣資料庫分析其中36個基因在本土肺腺癌病人組織中的表現。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。 經過一連串的分析，我們集中精力於兩組基因, Slit2及EMP2。這兩個基因的表現在同一個病人之正常組織及肺腺癌腫瘤中有非常顯著的差異表現量。我們正積極的進行基因功能的分析,主要的方法是利用RNA干擾及過度表現這些基因,以觀察這些基因可能扮演的生物功能。 Slit2在肺腺癌的表現顯著降低，我們因而想了解Slit2在肺癌的形成過程中所扮演的角色。因此,我們利用RNA干擾技術將slit2的表現剔除，觀察癌細胞失去slit2表現後的影響。初步的發現是,slit2的表現降低至原來的0.5%至10%時細胞的生長速度顯著的下降,使位於G2/M週期的細胞數增加。有趣的是當slit2被抑制之後,似乎對細胞與細胞之間的接觸產生很大的改變,這是我們接下來想研究的方向之一。除此之外,我們想看看Slit2的表現是否會影響細胞的移動及侵犯性,並進一步觀察slit2在老鼠體內對細胞的影響。 我們已將slit2的全長cDNA複製出來,由於我們目前沒有適當的Slit2抗體,我們將slit2接上不同蛋白,將來可利用這些蛋白的抗體偵測Slit2的表現。希望增加Slit2的表現,可以幫助我們釐清slit2 RNAi所產生的影響。 Slit2本身是一個約200Kd大的分泌型蛋白，它的已知受體為Robo1。Robo 家族裡除了Robo1外尚有Robo2, Robo3和Robo4。這些家族成員有非常高的相似性,我們初步的分析發現大部分的Robo成員於肺組織中有表現。由於slit2的生物功能多樣性,它很可能透過不同的受體進行不同的訊息傳遞。我們因而想探討在肺中,slit2所扮演的生物功能為何,並利用那一個受體進行訊息傳遞？
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, two subtractive cDNA libraries via suppressive subtractive hybridization were established. We developed a differential screening method to identify differentially expressed genes and 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. To investigate the expression patterns of these genes adenocarcinomas in Taiwan, we analyzed these genes on microarray database of lung adenocarcinomas via collaboration with Dr. Chi-Yin Huang (NHRI). 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. We are vigorously studying two candidate genes, Slit2 and EMP2. The expression of slit2 mRNA is down regulated in lung adenocarcinoma. Since slit2 exhibits different functions in different cell types, we set up to study the role of Slit2 in tumorigenesis in lung cancer. When slit2 mRNA is reduced to less than 10% of original level by RNA interference, the growth rate of the cell is greatly reduced. Interestingly, we found that interfere slit2 expression affecting cell-cell interaction. Since Slit2 affect cell motility in other systems, the migration ability will be examined in these cell lines with slit2-RNAi. We will inject cells into immune-deficient mice to study the effect of Slit2 in tumor formation in vivo. We also cloned full-length slit2 cDNA. At present, we are not be able to obtain good antibody to detect Slit2, therefore we generate different kind of fusion protein for detection its expression in cell lines. We hope over-expression of slit2 would help us to clarify the phenotypes observed in slit2-RNAi stable clones. The known receptor of Slit2 is Robo1. There are four Robos, Robo1, Robo2, Robo3 and Robo4, in the family. These family members share high level of identity in amino acid level. Our studies showed that these family members express in lung. Since Slit2 possesses complex cell |
