探討RpL37基因魚鱗狀上皮性鼻咽癌形成之功能分析

Abstract

鼻咽癌好發於東南亞國家，尤其是中國、香港、新加坡和台灣。其致病原因相當複雜，至今仍尚未被研究徹底。早期認為EB病毒和鼻咽癌的發生有很大的關係，但近期許多研究報導都認為EB病毒並非是鼻咽癌發生的主因而是促使鼻咽癌惡化的因素。因此，為了更深入的探討鼻咽癌癌化相關致病基因的分子機制，我們使用cDNA微陣列分析（cDNA microarray analysis）以比較正常鼻咽黏膜上皮細胞和鼻咽癌細胞之間表現的差異，再經由即時定量聚合酶連鎖反應（Quantitative RT-PCR）及西方墨點法（Western Blotting）發現SRY（sex-determining region Y）-box5（SOX-5）在鼻咽癌細胞中的表現有相當顯著的增加。進一步去研究SOX5基因高度表現的分子機制時我們發現Ribosomal Protein L37（RpL37）在鼻咽癌細胞中亦有較高的表現量。在實驗室所建立的鼻咽癌細胞株尤其在undifferentiated carcinoma NPC-TW01N1和NPC-TW06N1中的表現顯著較高。為了更進一步的去探討RpL37在鼻咽癌中扮演的角色以及和其他基因間相互調控的關係。我們發現在調控NPC中SOX5和RpL37的表現有正向關係，這也表示RpL37可能在NPC形成過程中扮演一個相當關鍵的角色。 為了研究RpL37在NPC中的功能我們使用搭載shRpL37的lentivirus去獲得一個穩定的NPC-TW01N1細胞株，我們發現在腫瘤細胞中RpL37的mRNA和蛋白質表現都有顯著的下降，而RpL37的下調可以對細胞的增生、爬行和穿透的能力有顯著的提升，除此之外也可以讓p53的表現隨著增加。就像大家知道的p53是一個可以影響細胞週期的抑癌基因，我們使用流式細胞儀去分析在用shRpL37處理過的細胞株細胞週期是否有改變。最後我們發現在使用shRpL37感染過的NPC-TW01N1細胞株較未處理過的細胞株在S期的細胞比例下降大約有28%，然而在帶有shRpL37處理的NOD/SCID小鼠中細胞生長和轉移的能力都有被顯著的提升，小鼠的體重在最後一個星期大幅的下降也讓我們在犧牲時發現小鼠的健康狀況不佳，可以看見在腸道的部分有大面積的潰爛。 由這樣的結果我們可以推論，RpL37基因在鼻咽癌的腫瘤生長中扮演一種會促使鱗狀上皮性鼻咽癌形成的因素並會使腫瘤生長速度顯著增加和移動、侵犯性能力的加強。

Nasopharyngeal Carcinoma (NPC) is one of the most common cancers among Chinese living in southern China, Hong Kong, Singapore and Taiwan. The cause of the disease is quite complex, and the molecular mechanisms involved in the pathogenesis of NPC still are not yet well defined. Although it was proposed that Epstein-Barr virus (EBV) is closely associated with NPC pathogenesis, but recently many studies have reported that EBV behaves more likely as progression factors but not initiation factors. The purpose of this research was to find out the genes associated with NPC pathogenesis. Using cDNA microarray analysis of mRNA expression between NPC cell lines and normal nasal mucosal epithelial cells, SOX5 gene expression was found significantly increased in NPC cell lines by quantitative RT-PCR and Western blot analysis. In our previous studies of the function of SOX-5 gene in NPC, we found that Ribosomal Protein L37 (RpL37) was also significantly increased in NPC cell lines, especially in NPC-TW01N1 and NPC-TW06N1 cell lines. To further identify the relationship between SOX5 and RpL37, we performed some investigation to clarify this condition. We found that SOX5 and RpL37 gene expressions could be reciprocally regulated in NPC cells and suggested that RpL37 may be a critical factor for the growth of NPC tumors. To study the role of RpL37 in the molecular pathogenesis of NPC and its functions, we used a stable shRpL37 lentivirus infected NPC-TW01N1 cell lines. We found that the expression of RpL37 mRNA and protein was downregulated remarkably in these call lines. This gene could promote tumor cell migration, proliferation and invasion in vitro. In addition, the down-regulation of RpL37 could up-regulate the expression of p53. We know that p53 is a suppressor gene which could affect the cell cycle. We used flow cytometry to analyze the difference between cell lines with or without shRpL37 infected. Finally, we found that the S-phase cells treated by shRpL37 lentivirus decreased about 28% than NPC-TW01N1 cell lines without infection. However, in NOD/SCID mice bearing shRpL37 infected NPC xenograft, the tumor growth and metastatic activity were moderately increased, and the weights of shRpL37 infected NOD/SCID mice were obviously decreased at last week. We also found those NOD/SCID mice were not healthy when sacrified, their intestinal tract showed focal ulceration and almost no stool in the large intestine. It is concluded that the downregulation of RpL37 gene may play a role to promote the formation of nasopharyngeal carcinoma of nasopharynx and as a suppressor gene in NPC pathogenesis to affect NPC migration, proliferation and invasion in addition to it chemotactic property.