頭頸部鱗狀細胞癌為全世界第六常見的癌症。口腔癌為頭頸部鱗狀細胞癌的一種,且在台灣位居男性十大癌症死因之第四位。在許多癌症中,已發現microRNA的失衡和癌症的發展及轉移有關。microRNA為一長約19~25個核甘酸構成的小片段不編碼的序列,藉由和標的基因mRNA上3’-UTR鹼基配對來調控標的基因後轉譯修飾的表現。我們實驗室利用microRNA microarray發現在口腔癌細胞株中的miR-99a表現量和正常口腔角質細胞比是下降的。進一步利用Real-time PCR也發現miR-99a的表現量在口腔癌細胞株和臨床病人檢體中皆是下降, 因此我們推論miR-99a在口腔癌可能扮演抑癌基因的角色。瞬間轉染 miR-99a或是在miR-99a 穩定表現的口腔癌細胞株中,皆可發現miR-99a可以抑制口腔癌細胞的爬行和侵襲能力；而miR-99a對於細胞增生的部分則因不同細胞株有不同的影響。我們也利用antagomiR-99a抑制miR-99a,發現可以促進口腔癌細胞的爬行和侵襲能力。運用microRNA 標的基因預測軟體我們找到4個miR-99a可能標的的下游基因。接著利用含有3’-UTR的冷光酶報導系統(luciferase reporter assay) 和定點突變(site-directed mutagenesis)證明miR-99a會藉由調控MTMR3(myotubularin related protein 3) 的3’-UTR明顯降低其冷光酶的活性。而在MTMTR3 的 3’-UTR中，miR-99a的 seed region發生突變情況下則冷光酶活性不變。半定量RT-PCR結果顯示miR-99a和MTMR3 mRNA表現量在口腔癌病人檢體中呈現反向關係，在口腔癌細胞株中則否。Western blot結果則顯示不管是內源性或是外源性，miR-99a和MTMR3 蛋白質表現量在口腔癌細胞株中皆呈反向關係。由於MTMR3在口腔癌細胞株中的角色尚未被說明，因此我們減弱內生性具有高表現MTMR3的 OC2。減弱OC2中的MTMR3可以明顯減弱口腔癌細胞株生長速率，爬行和侵襲Matrigel能力，由此可知MTMR3在口腔癌細胞株中扮演致癌基因的角色。由以上結果我們認為miR-99a在口腔癌細胞中扮演抑癌基因的角色，透過調控MTMR3表現量。未來我們還會繼續研究miR-99a和MTMR3的作用機轉和在口腔癌細胞中扮演的角色。

Head and neck cancer is the six most common carcinomas worldwide. Oral cancer, one of head and neck cancer types, is the fourth leading causes of male cancer death in Taiwan. The deregulation of certain microRNAs has been associated with the progression and metastasis of various cancer types. MicroRNAs are noncoding RNA molecules in a length of 19~25 nucleotides, which can mediate gene expression by base pairing to the 3’untralslated region (3’-UTR) of target mRNAs. Using human microRNA microarray analysis, we found that miR-99a was most down-regulated in oral cancer cells when compared to normal oral keratinocytes. Real-time PCR analysis further confirmed the down-regulation of miR-99a both in the majority of tested oral cancer cell lines and human clinical specimens. Therefore, we hypothesized that miR-99a might act as a tumor suppressor in oral cancer. Transient and stable expression of miR-99a inhibited the migration and invasion of oral cancer cells while having differential expression on cell proliferation depending on the cell types. Using antagomiR-99a to silence the effect of miR-99a enhanced cell migration and invasion. Four genes were predicted to be putative downstream targets of miR-99a. We then used 3’-UTR-bearing luciferase reporter assays and site-directed mutagenesis and confirmed that miR-99a could significantly decrease the luciferase activity regulated by the MTMR3(Homo sapiens myotubularin related protein 3) 3’-UTR but not the one with mutations in miR-99a seed region. Semi-quantitative RT-PCR analysis showed an inverse expression of miR-99a and MTMR3 mRNA in clinical specimens but not in oral cancer cells. Western blot analysis, however, showed the inverse relation of endogenous or exogenous miR-99a and MTMR3 protein expression in oral cancer cells. Since the role of MTMR3 in oral cancer has not been elucidated, knocking down MTMR3 expression in high MTMR3-expressing OC2 cells significantly impaired the ability of oral cancer cells to proliferate, migrate or invade Matrigel, suggesting an oncogenic role of MTMR3 in oral cancer cells. Together, our findings suggest that miR-99a acts as tumor suppressor through regulating MTMR3 expression in oral cancer cells. We will continue to study the mechanistic relationship of miR-99a and MTMR3, and the functional implication of this relation in oral cancer.

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