FZD1基因在鼻咽癌之功能分析

Abstract

鼻咽癌發病原因至今仍尚未被研究清楚，探討其原因分為三種─即環境因素、病毒及遺傳因素。鼻咽癌是中國南方、香港、新加坡及台灣等地的華人好發的癌症，過去普遍對於鼻咽癌的認識皆認為其與Epstein-Barr virus (EBV)的感染有極大的關聯性，但是近年來研究認為EBV並非造成鼻咽癌發生的原因，而與造成鼻咽癌惡化有關。本實驗研究的主要目的，就是要觀察與鼻咽癌致病相關的基因及其功能分析。在之前，我們實驗室曾建立十株鼻咽癌細胞株。我們使用cDNA微陣列分析（cDNA microarray analysis）去比較鼻咽癌細胞株及正常鼻咽黏膜上皮細胞株之間的表現差異，再經由即時定量聚合酶連鎖反應(Quantitative RT-PCR)及西方點墨法（Western Blotting）去研究而發現有很多基因在鼻咽癌細胞中的表現量有顯著異常的變化。我們從這些基因中挑出了FZD1基因來做本篇研究主軸，進而去探討FZD1在鼻咽癌細胞中所扮演的角色。我們發現FZD1基因在鼻咽癌細胞中的表現量有顯著的上升。然而，在40個鼻咽癌病人切片中也可以看到有52.5%有FZD1的高度表現。至今尚未有研究報導FZD1與鼻咽癌之間的關係，所以我們藉此進而深入研究FZD1對鼻咽癌的病理機理所佔的角色及其功能。首先，我們利用qRT-PCR分析正常鼻咽上皮細胞及鼻咽癌細胞株之間FZD1基因表現的差異，發現FZD1在NPC-TW01有高度的表現，此外，再利用免疫組織染色法及西方點墨法發現FZD1在不同鼻咽癌細胞株中蛋白表現量均有增加的現象。為了進一步研究FZD1對於鼻咽癌細胞株中所扮演的角色，我們利用干擾質體(shRNA)轉染鼻咽癌細胞株NPC-TW01，我們發現轉染的鼻咽癌細胞株可以顯著降低FZD1的mRNA和蛋白質表現。接著進而發現，其顯著的降低鼻咽癌細胞株的增生、轉移及侵襲力，而會增加鼻咽癌細胞的凋亡。在活體的動物實驗中，我們利用免疫不全小鼠(NOD SCID)，經由異種移植的實驗數據中發現，經由shFZD1轉染的鼻咽癌細胞(NPC-TW01)在第7週時，腫瘤的重量及體積皆顯著的降低。此篇研究發現FZD1與鼻咽癌的增生、轉移、入侵及凋亡有關聯性，這樣的發現對於未來在鼻咽癌的分子標靶治療具有一定的潛力。 在過去的研究報告中，發現有一些SOX家族可以與WNT訊號途徑有相互作用，在我們先前的研究中， SOX5在鼻咽癌細胞中也有高度的表現，而SOX5與FZD1之間的關係尚未被研究，透過這樣的發現，進而我們去探討SOX5與FZD1之間是否存在關聯性。在研究結果中發現，SOX5與FZD1之間存在著互相調控的關係。

NPC is one of the most common cancers among Chinese living in southern China, Hong Kong, Singapore and Taiwan. The pathogenesis of Nasopharyngeal Carcinoma (NPC) is still not well defined yet. Many investigators have prepared three possible factors for the pathogenesis of NPC, the environmental factors, virus infection factor and genetic factors. In the past, universally understanding of NPC is considered to have a great relevance with Epstein-Barr virus (EBV) infection. However, current studies have reported that EBV behaves more likely as progression factor but not initiation factors. The aim of this research is to find out the genetic attention of NPC. First of all, using cDNA microarray analysis of mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells, we found that the expression of many genes significantly increased in NPC cell lines by quantitative RT-PCR and Western blot analysis. According to the cDNA microarray data, we selected FZD1 gene and investigated its biological functions in NPC. FZD1 was significantly increased in NPC cell lines, especially in NPC-TW01N1 cell lines. And the result also can see in NPC patients’ biopsy specimens. In 40 cases of NPC patients’ biopsy, 52.5% had high expression of FZD1. The relationship between FZD1 and NPC is still not clearly reported. For the reason we used qRT-PCR and Western bloting to analyze the mRNA and protein levels of normal nasal mucosal epithelial cells and NPC. We found that both of mRNA and protein levels are significantly increased in NPC cell lines. To further identify the functional roles of FZD1 gene in NPC, we utilized FZD1 shRNA-lentiviral vector to knockdown the expression of FZD1 gene in NPC-TW01. In infected NPC-TW01 tumor cells, their expression of mRNA and protein levels were significantly decreased. This in turn resulted in a significant reduction in cell proliferation, apoptosis, migration and invasion. In vivo, in NOD-SCID mice bearing the NPC xenografts, the tumor weight and volume of the group of mice bearing lentivirus infected NPC-TW01 tumor cells were significantly decreased compared with the group of shLuc control. This study might suggest that FZD1 plays oncogenic role in NPC pathogenesis, including enhancing proliferation, migration and invasion. These novel findings might have potential implication in further molecule targeted therapy for NPC. In previous published studies, they found the interaction between SOX family and WNT signaling pathway. And in our previous study, we found that the expression of SOX5 is significantly higher in NPC than in NNM. The relationship between SOX5 and FZD1 has not be defined yet. Moreover, we investigated the relationship between SOX5 and FZD1. In the current result, there exists an interactional relationship between SOX5 and FZD1.