IFI6 基因在鼻咽癌腫瘤生長所扮演之角色

Abstract

鼻咽癌是種好發於鼻咽部的癌症 ，其發生的族群以東南亞國家為主例如：香港、新加坡、及中國沿海地區(廣東、福建)，此外非洲的人民也不少人得此病，世界衛生組織將鼻咽癌分為三類，第一類為：未分化的鱗狀上皮細胞癌、第二類為：非角質化細胞癌、第三類為：未分化細胞癌，鼻咽癌的致癌因素包括：病毒感染、飲食習慣、遺傳、環境..等。而其中最為大家所耳熟能詳的是EBV病毒感染，研究雖然顯示患有第一類未分化鱗狀上皮細胞癌的病人約有百分之96機率，可在他們的血液中測到EBV病毒的DNA，但根據許多報導顯示，我們有理由相信EBV不是導致鼻咽癌發生的主因，而是促使鼻咽癌增生的發生，本論文之主要研究目的，是想找出導致鼻咽癌發生的相關基因，首先我們使用cDNA 微陣列分析去比較正常鼻咽黏膜上皮細胞與鼻咽癌的表現量，再由定量聚合酶連鎖反應(Quantitative RT-PCR)、西方點墨法(Western Blotting)的研究發現chemokine (CC motif) of ligand28 (CCL28)基因，在鼻咽癌細胞中有明顯增加，而且在進而研究CCL28在鼻咽癌中扮演的角色及其造成的影響時，我們發現Interferon alpha-inducible protein 6 (IFI6)基因在鼻咽癌細胞中也有明顯增加的表現量，尤其是在TW06N1細胞上，因此我們決定深入研究IFI6基因在鼻咽癌中扮演的角色以及其功能，首先我們訂購IFI6之shRNA ，將其感染到鼻咽癌細胞中，建立一個knockdown的系統，使鼻咽癌細胞中的IFI6表現量下降，之後我們去做一連串的細胞功能分析(functional analysis)，發現鼻咽癌細胞不管在爬行能力、侵襲力、以 及細胞增長能力方面都有明顯下降的趨勢，而另外在細胞凋亡現象方面，我們也發現其凋亡現象增加，與此同時，我們也另外建立了另一個shCCL28之細胞株，一樣是用shRNA病毒去感染鼻咽癌細胞使其細胞中的CCL28的表現量下降，我們發現當CCL28的表現量下降時，IFI6的表現量也跟著下降，我們相信CCL28與IFI6之間可能存在一個調節的關係。在異種移植的鼻咽癌腫瘤 (NPC xenografts)長於NOD-SCID之老鼠的實驗中發現以抑制IFI6基因表現的shRNA所轉染的鼻咽癌細胞在NOD-SCID老鼠體內長到第六週時，腫瘤重量比控制組小了百分之七十八，在免疫染色與組織病理分析時發現抑制IFI6基因表達能減緩異種移植的腫瘤細胞生長並伴隨著嚴重的細胞凋亡。基於以上這些實驗結果 我們相信IFI6基因在未來很有潛力成為新的鼻咽癌治療標的。

Nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. It is one of the most common cancers among East Asia (Taiwan,Hong Kong , Singapore,southern China) and Africa . The World Health Organization (WHO) criteria for nasopharyngeal cancer has been published. They are Type 1 .Differentiated squamous cell carcinoma , Type 2 .Non keratinizing carcinoma,Type 3. Undifferentiated carcinoma. Nasopharyngeal carcinoma is caused by a combination of factors including viral infection , environmental influences, and heredity. The viral influence is associated with infection with Epstein-Barr virus , EBV DNA was detectable in the blood plasma samples of 96% of patients with non-keratinizing NPC. The purpose of this research was to find out the genes associated with NPC pathogenesis . First , we use cDNA microarray analysis of mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells,CCL28 gene expression was found significantly increased in NPC cell lines by quantitative RT-PCR and IHC. In our previous studied of the function of CCL28 gene in NPC , we found that Interferon alpha-inducible protein 6 ( IFI6 ) was also significantly increased in NPC , especially in NPC-TWO6N1. To further identify the relationship between CCL28 and IFI6 , we performed some investigation to clarify this condition. We found the CCL28 could affect the gene expression of IFI6 . In order to study the role of IFI6 in the molecular pathogenesis of NPC and its functions，we transfect shRNA in NPC cell to construct stable shIFI6 NPC cell lines as a knockdown system . we found the tumor cells could be down-regulated to express IFI6 mRNA and protein about 40-50% , We also found that when IFI6 was down-regulated , the ability of cell migration , invasion ,proliferation were decreased . On the other hand , the apoptosis of NPC was promoted. We also knocked down CCL28 by using shRNA to create a shCCL28 system and found that when CCL28 was down-regulated , the protein expression of IFI6 was decreased . However , when IFI6 knockdown , the gene expression of CCL28 was not significantly changed. In vivo , we have injected shIFI6 NPC cell line to NOD-SCID mice for 6 weeks , the weight of tumor diminished about 78% and tumor size decreased about 55% after 6 weeks , accompanied by necrosis and apoptosis in the NPC-TW06N1 xenografts treated with IFI6 shRNA histopathologically. These novel findings of the functional role of IFI6 may have a potential implication in molecular target therapy for NPC.