NOLC1與P53協同調控Bax基因表現及其調控鼻咽癌腫瘤生長之機制

Abstract

鼻咽癌好發於東南亞國家。雖然早期EB病毒被認為和鼻咽癌的發生有很大的關係，但是近期許多的研究報導都認為EB病毒並不是導致鼻咽癌發生的原因而是促使鼻咽癌惡化的因素。因此，有關於鼻咽癌癌化的分子機制仍然需要被深入探討。為了探討有關鼻咽癌的分子機制，先前我們實驗室用利用PCR-SelectTM cDNA相減雜交的方式及基因晶片分析比較正常的鼻咽細胞和鼻咽癌細胞之後發現NOLC1基因的表現量高於正常的鼻咽細胞。而後，我們發現NOLC1基因和TP53基因協同調控MDM2的基因表現。為了更深入探討是否NOLC1基因和TP53基因協同調控更多TP53調控的基因，我們用lentiviral shRNA transfection系統干擾NOLC1基因的表現量，藉由qRT-PCR及西方墨點法皆發現受TP53調控的細胞凋亡基因Bax也受NOLC1基因調控，接著再利用螢光素酶檢測法發現NOLC1調控Bax的promoter位置也正好是TP53調控Bax的promoter位置。接著我們在讓鼻咽癌短暫高量表現NOLC1基因也發現Bax基因有微量上升，但是其蛋白量沒有顯著上升，這表示其中勢必有其他因子干擾Bax基因的上升。深入探討NOLC1蛋白及TP53蛋白的關係，我們發現這兩個蛋白並沒有互相結合。接著，我們觀察NOLC1基因本身對整個鼻咽癌細胞株的影響發現在NOLC1基因短暫高量表現後，利用TUNEL assay檢測到細胞凋亡的細胞減少了，但是在看MTT assay後並沒有觀察到細胞生長速度的增加。然而我們用cDNA microarray卻觀察到在當NOLC1基因表現上升後，很多致癌基因表現量上升另外也有很多抑癌基因表現量下降，這表示NOLC1基因在鼻咽癌細胞株勢必扮演一個促使鼻咽癌細胞癌化的因素。

Nasopharyngeal Carcinoma (NPC) has higher incidence in Southeastern Asia as compared to other populations in the world. Although it was proposed that Epstein-Barr virus (EBV) is closely associated with NPC pathogenesis, many studies showed that EBV behaves more likely as an enhancer but not an initiator. Thus, the molecular mechanisms involved in the pathogenesis of NPC remained to be fully analyzed. Previously, using PCR-selectTM cDNA subtractive hybridization with microarray analysis our laboratory has shown that NOLC1 gene expression was upregulated in most NPC cell lines. Moreover, NOLC1 acts as an enhancer to coactivate with TP53 to regulate MDM2 expression. In order to investigate whether this is a general phenomenon in most TP53- regulated genes, in this study, lentivirus transfection system was used to establish stable NOLC1- knockdown NPC cell lines. By using quantitative real time PCR and Western blotting, we found that when NOLC1 was downregulated, the expression of TP53- regulated apoptosis gene, such as Bax, was also inhibited. The luciferase assay further supported that NOLC1 could regulate Bax promoter activity at its p53-responsive promoter region. Luciferase assay also showed that TP53 requires NOLC1 for coactivation of Bax expression. In other words, NOLC1 cooperates with TP53 to regulate the Bax expression is similar as MDM2 expression regulated by both genes. However, when NOLC1 was transiently overexpressed in NPC cell lines, we found modertate increase of Bax mRNA expression and but did see upregulation of Bax protein expression, indicating that the proliferating NPC cells may have other mechanism to suppress or enhance degradation of Bax protein. This result suggests that NOLC1 plays a role as an enhancer to cooperate with PT53 to regulate PT53-regulated target Bax gene expression. However, co-immunoprecipitation of Western blot analysis and NOLC1 and p53 colocalization of both proteins did not suggest these two proteins can bind together. Furthermore, we analyzed apoptosis behavior after NOLC1 overexpression by TUNEL assay. We found that overexpressed NOLC1 resulted in fewer apoptotic cells. However, proliferation rate is not enhanced after NOLC1 overexpression. Also, cDNA microarray analysis of NOLC1 overexpressed NPC cells showed a group of oncogenes upregulated and some oncosuppressor genes downregulated, suggesting that NOLC1 may also play some role in regulation o NPC tumorgenesis. In conclusion, NOLC1 not only can co-activate with TP53 to regulate the oncogene MDM2 expression but also the Bax gene in NPC pathogenesis.