肺癌為十大癌症之一，在世界衛生組織的統計下，每年約有140萬人死於肺癌。而且合併計算其它併發症的病人，肺癌的五年存活率相較低於其他癌症只有15.2%。Nucleolin在細胞具有許多的功能，包含了調控細胞增生、細胞凋亡、信使RNA (messenger RNA)的穩定性等。先前的研究已經指出nucleolin在腫瘤細胞中大量表現，若抑制nucleolin的表現會影響到癌細胞增生，顯示出nucleolin在癌化過程中扮演重要角色。而在過去實驗室利用組織免疫染色法觀察肺腺癌病患的肺組織nucleolin的表現，結果顯示在組織中的nucleolin有不同的表現量，經統計分析發現若nucleolin表現量較高的病患其預後較差相較於低nucleolin表現的病患;另外利用西方墨點法觀察肺腺癌病患肺組織和實驗室建立的EGFR (exon19 deletion)和Kras活化誘導肺癌轉殖鼠組織中nucleolin的表現，而實驗結果顯示出不論在肺癌病患組織或是實驗室建構的誘導肺癌轉殖鼠組織中c-nucleolin (55 KDa)有非常顯著性增加。根據結果我們假設c-nucleolin會參與肺癌細胞轉移過程當中。但是我們非常好奇在肺癌進展過程中c-nucleolin大量累積是扮演什麼角色以及是誰去切nucleolin和切位位於何處。為了證實這個假說，首先我們建構GFP-Nucleolin載體，根據結果推論出c-nucleolin切位為nucleolin第251到261個胺基酸，之後更進一步架構GFP-nucleolin-myc質體來確認確切切位是位於這區間哪一個胺基酸。利用架構好的質體進行單點突變試驗分別將這區間的胺基酸單一進行點突變，發現在255這個胺基酸位置若突變會使c-nucleolin (55KDa)切下量明顯減少許多，結果證明nucleolin切成c-nucleolin的位置是在255這個胺基酸位置。另外方面我們好奇的是哪個酵素去切nucleolin形成c-nucleolin，為了要找出是哪個酵素去切nucleolin，在尋找可能會切nucleolin的酵素中發現基質金屬蛋白酶會切nucleolin，於是更進一步使用小髮夾RNA （Short hairpin RNA， shRNA）分別降解不同的基質金屬蛋白酶表現，結果發現若降解基質金屬蛋白酶7 的表現會影響c-nucleolin的表現量。而在確認切位是位於何處以及切 nucleolin的酵素後，我們接著探討這c-nucleolin片段在肺癌中扮演什麼角色，是否會更加影響細胞生長速率和轉移能力。利用我們建構的c-nucleolin片段 (255-710)的載體轉染至肺癌細胞發現到會增進癌細胞生長速率並且顯著性的增加細胞轉移及侵犯能力。根據實驗結果，我們好奇的是c-nucleolin增強癌細胞轉移侵犯能力是透過影響哪些基因造成的，下一步利用DNA微陣列晶片分析nucleolin和c-nucleolin是影響到哪些基因表現，其中c-nucleolin影響的基因中和癌症進展相關有基質金屬蛋白酶 9 (Matrix metalloproteinases 9)、ALK (Anaplastic lymphoma kinase)、CRTC1 (CREB-regulated transcription coactivator 1)…等基因，其中基質金屬蛋白酶 9最引為注目，過去許多研究指出基質金屬蛋白酶 9與癌症轉移等相關，在這之中發現c-nucleolin會結合上基質金屬蛋白酶 9的3’ UTR區域並更增加基質金屬蛋白酶 9穩定性，使基質金屬蛋白酶 9大量表現造成癌細胞轉移，使癌症進展更加惡化。而綜合以上結果，證實當腫瘤持續惡化時 nucleolin被基質金屬蛋白酶 7切割成 c-nucleolin並大量累積，促使癌細胞增生並且增強癌細胞轉移的現象。

Nucleolin distributes in the nucleolus, nucleus and cytoplasm of whole cell contains multifunction such as ribosome maturation, cell growth, apoptosis, mRNA regulation, pre-rRNA transcription. Previous studies indicated that overexpression of nucleolin was found in the cancer cells, and knockdown of nucleolin could inhibit cell proliferation, indicating nucleolin might be related to tumorigenesis. Interestingly, our recent results reveal that not only accumulation of nucleolin was found in lung cancer formation, but nearly all of the nucleolin was modified by post-translational processing into a cleavage form, 55 KDa, in animal and clinical samples. Therefore, cancer cells how to accumulate and cleave nucleolin, and what is the role of this overexpressed truncated nucleolin are interesting and need to be elucidated further. Our recent study has known that activation of Kras and EGFR contributes to nucleolin overexpression and Sp1 involves in nucleolin expression. In this study, we will try to clarify the mechanism and function of nucleolin cleavage. By using the truncated and single nucleotide mutation experiments, we found that nucleolin was major cutted at Asp 255th. Furthermore, various proteases inhibitors used here to block the nucleolin cleavage found that Marimastat, Matrix metalloproteinases (MMPs) inhibitors, can prevent the nucleolin cleavage, implying MMPs might be involved in the nucleolin cleavage. Different shRNA of MMPs used to knockdown MMPs individually found that knockdown Matrix metalloproteinase 7 (MMP7) could reduce the nucleolin cleavage obviously, indicating that MMP7 could affect the nucleolin cleavage. Finally, by using the truncated nucleolin to study its effect in cancer cell proliferation and metastasis found that truncated nucleolin increased the cell proliferation and metastasis activity strongly through increase in the oncogenes expression including mmp9, alk and so on. Taken together, nucleolin cleaved by MMP7 at Asp 255th increases oncogenes expression, leading tumor growth and metastasis.

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