探討微小核醣核酸17-92群簇對人類口腔鱗狀上皮細胞癌移行能力之影響

Abstract

口腔癌之普及率在全球常見癌症中排名第八位，且經行政院衛生署公布之統計資料，民國九十八年與九十九年的十大癌症死因中，口腔癌是唯一排名上升的惡性腫瘤(第六升至第五)，足以顯現口腔癌在台灣的盛行率與研究潛力。約有九成的癌症病患最後死於癌症轉移的發生，為了提升癌症病患的存活機率，關於癌症轉移的研究已經成為刻不容緩的議題。本實驗室為了釐清口腔癌發生轉移的相關分子機制，利用了miRNA的微陣列對TW2.6與TW2.6 MS-10進行分析，發現一個miRNA群簇－miR-17-92 cluster，在移行能力較高的TW2.6 MS-10細胞株中有下降的現象。於是我們進一步分析常見口腔癌細胞株的miR-17-92 cluster表現量，發現其表現量與各細胞間的移行能力呈現負相關。為了確認miR-17-92 cluster是否能影響細胞移行能力，我們將miR-17-92 cluster在TW2.6 MS-10與SAS中過量表現，發現其確實能降低此兩細胞株的移行能力。而為了瞭在此cluster中，何者扮演主要調控者的角色，我們又分別在TW2.6 MS-10與SAS中分別過量表現miR-17、miR-19b、miR-20a與miR-92a，結果發現只有miR-17與miR-20a對兩株癌細胞具有明顯的移行抑制能力。而從病人的數據也顯示，在高期數與發生淋巴轉移的病人，miR-17與miR-20a有比較低的表現量。從臨床檢體與細胞實驗中顯示，在口腔癌中miR-17與miR-20a似乎扮演著重要角色。為了釐清miR-17與miR-20a抑制細胞移行能力的分子機制，我們先以生物資訊軟體TargetScan與Microcosm進行下游標的的預測，並且利用Ingenuity Pathway Analysis (IPA) 對預測標的進行功能性的分析，篩選出同時是miR-17與miR-20a的下游標的，且對細胞移行能力有影響的目標基因－ITGβ8。經由RT-PCR發現，在TW2.6 MS-10細胞中ITGβ8比TW2.6有較高的表現量，但由暫時轉染各個miRNA的數據顯示，ITGβ8才是miR-17與miR-20a可能的下游基因。而為了確定ITGβ8在口腔癌中的功能，我們利用shRNA的方式抑制TW2.6 MS-10細胞中ITGβ8的表現，發現細胞移行能力隨著ITGβ8表現量下降而降低。接下來我們構築了野生型與突變型的ITGβ8 3’UTR來證明ITGβ8真的為miR-17與miR-20a的直接下游。實驗結果顯示，ITGβ8的確是miR-17與miR-20a的下游調控基因。綜合以上實驗結果顯示，miR-17-92 cluster確實有抑制口腔癌移行能力的功能，而其中又以miR-17與miR-20a為主要調控者，乃藉由種源序列與ITGβ8的3’UTR結合，使ITGβ8的mRNA進行降解而達到抑制細胞移行能力的效果。

Metastasis is always an important concern in oral squamous cell carcinoma (OSCC) treatment, and migration is the first and crucial step in this process. To investigate this issue, we established a more aggressive cell line-TW2.6 MS-10 by transwell selection from low-migration ability oral cancer cell line: TW2.6. To better understand the effect of microRNAs (miRNA) profile in OSCC metastasis, the miRNA microarray analysis between TW2.6 and TW2.6 MS-10 was performed. According to the array data, a miRNA cluster miR-17-92, including miR-17, miR19b, miR-20a, miR-92a, was significantly down-regulated in TW2.6 MS-10 compare to the parental cells. To further confirm whether this cluster directly regulates the migration ability in OSCC cells, miR-17-92 cluster was over-expressed in TW2.6 MS-10 and SAS cells. The results showed that the migration abilities were remarkably suppressed compared to control vector clones (P < 0.01, P < 0.01, respectively). In order to figure out which miRNA(s) in this cluster is the dominant regulator(s) in OSCC migration instruction, we cloned miR17, miR19b, miR-20a and miR92 separately and found that miR-17 and miR-20a of miR-17-92 cluster seemed to play a key role in this migration regulation (P < 0.01).The clinical data also supported this finding. Cross matching by TargetScan and Microcosm to predict the suspicious downstream target(s) of miR-17 and mir-20a in this system, a possible downstream target ITGβ8 was found. Taken together, here we first defined a new pathological role of miR-17-92 cluster as a tumor suppressor miRNA in OSCC migration regulation machinery.