清細胞型腎細胞癌相關基因之研究： I. 分析清細胞型腎細胞癌之全長cDNA庫 II. MYC活化於清細胞型腎細胞癌之致癌角色 III. 菸鹼醯胺-氮-甲基轉移酶在清細胞型腎細胞癌之癌化角色

Abstract

腎細胞癌佔世界上癌症的3 %，有逐年增加的趨勢，其中清細胞型腎細胞癌（ccRCC）是最常見的腎細胞癌亞型。由於腎細胞癌具有高轉移能力與高抗藥性，導致腎細胞癌病人有較差的五年存活率。 為了找出ccRCC中癌化相關基因，我們利用oligo-capping法建立ccRCC和正常腎臟的全長互補去氧核醣核酸資料庫。在進行去氧核醣核酸定序後，分別在ccRCC和正常腎臟中鑑定到4,356個基因與3,055個基因。藉由比較ccRCC與正常腎臟的基因表現圖譜，找到了201個表現上升基因與182個表現下降基因。我們以Q-PCR進一步證明20個未曾被發現在ccRCC中表現有變化的基因在ccRCC組織中有異常表現。 為了探討在ccRCC中異常的細胞訊息路徑，我們對383個ccRCC相關基因進行功能性網絡分析，並發現MYC路徑在ccRCC中是活化的。我們進一步證明MYC與MYC標的基因BCL2、CCND1、PCNA、PGK1和VEGFA的表現量有正相關。若抑制MYC表現，MYC標的基因的表現也會因此減少，且MYC也被證明會結合在BCL2、CCND1、PCNA、PGK1和VEGFA的基因啟動子上。此外，利用MYC siRNA減少MYC表現量可明顯抑制ccRCC細胞的增生、非固著依賴性細胞生長與細胞週期，這些結果證明了MYC活化對ccRCC的重要性。 菸鹼醯胺-氮-甲基轉移酶（NNMT）在ccRCC中是表現明顯上升基因之一。我們進一步研究NNMT在癌細胞中的功能，並發現具侵犯性的腎細胞癌細胞株中NNMT與基質金屬蛋白酶-2（MMP-2）的表現比不具侵犯性的腎細胞癌細胞株高，而且NNMT siRNA可明顯抑制腎細胞癌細胞株中MMP-2的表現。此外，在腎細胞癌組織中，NNMT和MMP-2的表現量有正相關，證明在腎細胞癌中NNMT與MMP-2表現有關連性。另外，我們發現NNMT酵素活性可能與引起MMP-2表現無相關性，而MMP-2基因啟動子上的SP1結合處、轉錄因子SP1和PI3K/AKT訊息路徑與MMP-2的表現有關。此外，實驗證明SP1與MMP-2基因啟動子的結合受到PI3K/AKT訊息路徑所調控。四個可能與NNMT有交互作用的蛋白也被鑑定出來。更進一步地，我們研究MMP-2在NNMT引起的細胞侵犯之角色，並發現MMP-2抑制劑或中和抗體均能抑制NNMT大量表現的細胞之侵犯能力；而在腎細胞癌細胞株中NNMT siRNA對侵犯能力的抑制作用可被MMP-2活性蛋白所回復，這些結果證明MMP-2對於NNMT引起的細胞侵犯是必要的。並且， NOD-SCID小鼠實驗證明NNMT的表現於腎細胞癌細胞株的生長與肺臟轉移扮演著重要角色。除此之外，我們也發現NNMT增加腎細胞癌細胞對抗癌藥物cisplatin所引起之細胞凋亡的抵抗力，其機制可能是NNMT的表現能抑制caspase 9/caspase 3/PARP的凋亡訊息與cisplatin引起的p53乙醯化。這些結果證明了NNMT在腎細胞癌中扮演重要角色。

Clear cell renal cell carcinoma (ccRCC) is the major subtype of RCC, accounting for 3% of malignancies worldwide with increasing incidence. ccRCC is characterized by its high invasive potential and insensitivity to traditional anti-cancer therapies, resulting in a poor five-year survival rate of patients with RCC. To investigate the genes associated with carcinogenesis in ccRCC, the full-length enriched cDNA libraries of ccRCC and normal kidney tissues were constructed by oligo-capping method. After nucleotide sequencing, 4,356 and 3,055 genes were identified from ccRCC and normal kidney, respectively. By analyzing gene expression profiles, 201 upregulated and 182 downregulated genes were identified in ccRCC. The differential expression of 20 ccRCC-associated genes, which have not been reported previously in ccRCC, were further demonstrated by quantitative real-time PCR. To study the deregulated cellular pathways in ccRCC, functional network analysis was performed using 383 differentially expressed genes. The signature of MYC pathway was found to be activated in ccRCC. The correlation between the expression levels of MYC and MYC-target genes, BCL2, CCND1, PCNA, PGK1 and VEGFA was demonstrated in ccRCC tissues. Moreover, our results showed that MYC siRNA suppressed the expression of the MYC-target genes, and MYC was associated with the promoter regions of these MYC-target genes in ccRCC cells. Furthermore, the knockdown of MYC expression significantly inhibited the uncontrolled proliferation, anchorage-independent growth and cell cycle progression of ccRCC cells. These results suggest an essential role of MYC activation for ccRCC. Nicotinamide N-methyltransferase (NNMT) was identified as one of the most upregulated genes in ccRCC. We found that the invasive ccRCC cell lines (786O and Caki-1) exhibited a higher expression of NNMT and matrix metalloproteinase-2 (MMP-2) than those of primary ccRCC cell lines. NNMT siRNA remarkably inhibited MMP-2 expression in ccRCC cells. Moreover, a positive correlation was identified between NNMT and MMP-2 expression in ccRCC tumors, suggesting that NNMT is associated with MMP-2 expression. Interestingly, NNMT catalytic activity may not be essential for NNMT-mediated MMP-2 expression. We further demonstrated that SP1-binding elements of MMP-2 promoter, transcription factor SP1 and PI3K/AKT pathway are involved in MMP-2 expression induced by NNMT. Moreover, the results revealed that the binding of SP1 on MMP-2 promoter is regulated by PI3K/AKT pathway. Four potential NNMT-interacting proteins were identified by yeast two-hybrid assay using human kidney cDNA library as prey. Furthermore, MMP-2 activity was demonstrated to be required for NNMT-mediated cell invasion. The in vivo experiment further indicated that NNMT expression plays an important role for the tumor growth and pulmonary metastasis of 786O cells in NOD-SCID mice. Additionally, the effect of NNMT expression on the resistance to anti-cancer drug was investigated. NNMT was found to increase the resistance to the apoptosis induced by cisplatin-mediated DNA damage in HEK293 cells and 786O cells. The results showed that the caspase 9/caspase 3/PARP apoptosis signaling was suppressed in NNMT-overexpressing cells. Moreover, the acetylation of p53 induced by cisplatin, which is a crucial signal to trigger apoptosis, was significantly reduced by NNMT expression. These results elucidate the cancer-promoting functions of NNMT in ccRCC.