2013-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/704700摘要：微小核醣核酸（microRNA）為一群不進行蛋白質轉錄的 RNA，可以調節數以百計的標的基因，並調控許多生物功能。之前，為了研究非小細胞肺癌對 EGFR tyrosine kinase inhibitor（TKI）產生抗藥性的機轉，我們將原本對 gefitinib 敏感的細胞株 HCC827，持續暴露在藥物濃度逐漸增加的 gefitinib 中，而篩選出對 gefitinib 具有抗藥性的細胞株HCC827/gef。有趣的是，此一對 gefitinib 具有抗藥性的細胞株 HCC827/gef, 不論是其細胞外型或相關生物標記，都明顯出現上皮 -間質轉化（ epithelial-to-mesenchymal transition，EMT）的現象。我們亦利用此 HCC827/gef 的細胞模型，以 microRNA 微陣列篩選與續發性抗藥性與上皮-間質轉化相關的 microRNA。其中，miR-503 為可能的標的。miR-503 在 HCC827/gef 細胞的表現，相較於具 EGFR TKI 感受性的 HCC827 親代細胞下降約三倍左右。我們發現將 HCC827/gef 細胞活化 miR-503 可以減弱上皮-間質轉化，以及與上皮-間質轉化相關的細胞行為（如細胞活動性與侵犯性），顯現了 miR-503在肺癌細胞生物學中尚不為人知的上皮-間質轉化調節者的功能。 近來，腫瘤幹細胞（cancer stem-like cell）在癌症生物學已引起了廣泛的注意。尤其，腫瘤幹細胞被認為影響了腫瘤的生成、復發、侵犯及轉移，並且在藥物抗藥性中扮演了重要的角色。同時，上皮-間質轉化與腫瘤幹細胞之間的關連在最近也得到許多討論。有一些證據顯示上皮-間質轉化的細胞擁有腫瘤幹細胞的特徵，而相對的腫瘤幹細胞也具有上皮-間質轉化細胞的生物型態。尤其許多調控上皮-間質轉化的訊號傳遞路徑，如Wnt、Notch、與 Hedgehog，也會促進腫瘤幹細胞的自我再生與維持。在最近的一篇研究中，發現肺癌細胞 ALDH1A1（腫瘤幹細胞標記之一）的表現與 EGFR TKI 的抗藥性有關。我們最近也發現 miR-503 的過度表現可以減少表現 ALDH1A1 的細胞族群，顯示miR-503 可能涉及腫瘤幹細胞的自我再生與維持的調控。此一計畫的目的在於探討miR-503 在調控肺癌幹細胞的角色，以及其對 EGFR 抑制藥物抗藥性的影響。我們也將尋找由 miR-503 調控，而與腫瘤幹細胞相關的標的基因。<br> Abstract: MicroRNAs, a class of non-protein coding RNAs, can post-transcriptionally regulate hundreds of their target gene and control a wide range of biological functions. Recently, to study the mechanism of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer, we have developed a cell-based system by culturing gefitinib-sensitive HCC827 cells in the presence of increasing concentration of gefitinib and were able to isolate resistant cell line HCC827/gef. Interestingly, this gefitinib-resistant cell line obviously has the transition of mesenchymal phenotype (epithelial-to-mesenchymal transition, EMT), as revealed by the morphological feature and EMT markers. To explore the role of microRNA in the process of EGFR-TKI resistance and EMT, we screened the microRNAs which are differently expressed with the development of EGFR-TKI resistance in the HCC827/gef cell model by microRNA low density array. Of the screened microRNAs, miR-503 represents one of the candidate, which is 3-fold down-regulated in HCC827/gef cells compared with the parental cells. In our recent work, we found that over-expressing of miR-503 in lung cancer cells attenuated EMT and EMT-related events such as cell migration and invasion, disclosing the unrevealed role of microRNA as a regulator of EMT in lung cancer biology. Recently, the cancer stem-like cell (CSC) has drawn much attention in cancer biology. Importantly, CSCs may contribute to carcinogenesis, tumor recurrence, invasion and metastasis, and play a key role in chemoresistance. Furthermore, a relationship between EMT and CSCs has recently emerged with evidence suggesting that EMT cells have cancer stem cell-like features and CSCs exhibit a mesenchymal-like phenotype. It is also known that many signaling pathways, such as Wnt, Notch and Hedgehog that regulate EMT, also drive CSCs self-renewal and maintenance. In a recent study, the association of ALDH1A1 expression, a putative marker of CSCs, with EGFR-TKI resistance in lung cancer cells was explored. We recently found that over-expression of miR-503 decreases the population of cells expressing ALDH1A1, as assessed using the commercial reagent ALDEFLUOR (STEM- CELL Technologies Inc, Vancouver, BC, Canada) and flow cytometry, suggesting that miR-503 is also involved in the regulation of renewal and maintenance of CSCs that are potentially associated with resistance to EGFR blockade. This project aims to disclose the role of miR-503 on the regulation of maintenance of lung CSCs, as well as its influence on resistance to EGFR inhibitors, and characterize the putative genes suppressed by miR-503 that are involved in this process.非小細胞肺癌EGFR tyrosine kinase inhibitor （TKI）抗藥性微小核醣核酸miR-503腫瘤幹細胞EGFR tyrosine kinase inhibitor (TKI) resistancemicroRNAmiR-503cancer stem-like cell