- 學位論文
研究EMP1和EMP2基因對人類肺癌細胞株的影響
Study the effect of EMP1 and EMP2 gene in human lung cancer cell line
摘要
中文摘要 PMP22為具有四個transmembrane domains 之穿膜蛋白，。目前己確認的5個PMP22的家族基因成員為PMP22、EMP1、EMP2、EMP3、LIM2。從本實驗室建立之精簡雜交基因庫中發現了N21 clone，N21基因在正常肺組織與肺腫瘤組織間有明顯的差異性表現量。經過一連串的實驗，我們認為N21與EMP2來自同一個轉錄產物。EMP2基因在肺癌病人之正常肺組織的表現量較肺腫瘤組織高約2~162倍。除此之外，其它的PMP22成員在肺腫瘤組織中均有較低的表現量。PMP22、EMP1、EMP3基因在肺癌病人之正常肺組織的表現量較肺腫瘤組織高，分別為2~45、2~89、1~25倍不等。我們選擇EMP1和EMP2做進一步的研究，觀察此二基因對於CL1系列細胞株的影響。CL1細胞株經體外篩出具有不同侵犯能力的亞株,依序為CL1-5＞CL1-1＞CL1-0，另外F4為CL1-5經體內篩選出具有高轉移能力之亞株。EMP1基因於高轉移之CL1-5、F4細胞中的表現為CL1-0與CL1-1之64至128倍。但是當EMP1 shRNA穩定表現在EMP1表現量較高的CL1-5、F4細胞中，發現EMP1並不影響細胞的遷徙能力。降低細胞中EMP1的表現對CL1-5和F4細胞生長有相反的影響； CL1-5細胞生長速度變慢了，但F4細胞生長速度增快了。抑制EMP1基因的表現時，會增加CL1-5細胞對化療藥cisplatin的敏感而造成細胞的死亡。當EMP2 shRNA穩定的表現在EMP2表現量較高的CL1-1細胞中，使得CL1-1細胞生長速度變慢，經由流式細胞儀分析發現EMP2受抑制時細胞有凋亡的現象。EMP2表現量降低時，會減少群落形成的能力。本研究的結論是EMP1及EMP2對細胞的生長有影響，但對不同的細胞有相反的影響；EMP1及EMP2對細胞的移動沒有影響；降低EMP2的表現會導致細胞調亡，降低EMP1不會；但降低EMP1的表現會增加細胞對cisplatin的敏感度。
關鍵字
精簡雜交基因庫並列摘要
The PMP22 family members encode tetra-span membrane proteins. Five family members are identified including PMP22, EMP1, EMP2, EMP3, and LIM2. The physiological roles of this family are largely unknown. Previously, a cDNA clone, named N21, was identified from a subtractive cDNA library that enriched genes with preferential expression levels in normal lung tissue. After a series of analyses we believe that N21 and EMP2 are transcribed from the same promoter. N21 and EMP2 showed correlated differential expression patterns between normal lung and lung cancer tissues. The mRNA level of EMP2 in normal lung was 2-162 fold higher than that in lung cancer. Other PMP22 family members also showed repressed gene expression in lung cancer; the mRNA levels of PMP22, EMP1, and EMP3 are 2-45 fold, 2-89 fold, and 1-25 fold higher in normal lung tissue than that in lung cancer, respectively. The mRNA levels of these PMP22 genes were also examined in various lung cancer cell lines. EMP1 showed notable expression patterns in CL1 series cell lines, which were selected with different in vitro invasive abilities, CL1-5>CL1-1>CL1-0, while F4 was derived from CL1-5 with highly invasive ability in vivo. The expression levels of EMP1 in CL1-5 and F4 are 64 – 128 folds higher than CL1-0 and CL1-1 cell line. We selected EMP1 and EMP2 for further study. To investigate the role of EMP1 and EMP2 in lung cancer, RNAi technique was applied to interfere EMP1 and EMP2 respectively to examine the effect of these genes in cell growth, cell death, cell invasive ability, and colony formation ability etc. When cells stably express EMP1 shRNA in CL1-5 and F4 cells, EMP1 showed oppositive effects on growth rate; reducing the expression of EMP1 decreased the growth rate of CL1-5 cells while increased the growth rate of F4 cells. Although EMP1 showed higher expression level in highly invasive cell lines, CL1-5 and F4, interfere EMP1 expression in these cell lines did not change their invasive abilities. However, decrease in the expression of EMP1 in CL1-5 cells will increase chemo-sensitivity to cisplatin. When EMP2 was interfered in CL1-1 cells decreased the growth rate, and induced cell death examined by flow cytometry. Down regulation of EMP2 also decreased cell attachment and colony formation abilities.
並列關鍵字
RNAi technique參考文獻
延伸閱讀
- 許智傑（2007）。探討Epithelial Membrane Protein 2基因對人類肺癌細胞株生長與侵犯能力之影響〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2007.00110
- 蔡佳螢（2008）。探討TGF-beta-induced factor homeobox 2基因對人類肺癌細胞生長及轉移的影響〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2008.00079
- 蔡孟軒（2005）。小蘗鹼抑制肺癌細胞生長之分子作用機轉之研究〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://doi.org/10.6834/CSMU.2005.00051
- 陳宛君（2012）。第二型轉麩胺酶對肺癌細胞存活和侵入的影響〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0003-1302201218330800
- Johnson, J. L., Pillai, S., & Chellappan, S. P. (2012). Genetic and Biochemical Alterations in Non-Small Cell Lung Cancer. Biochemistry Research International, 2012(), 719-736. https://doi.org/10.1155/2012/940405