口腔鱗狀細胞癌 (OSCC) 是全球常見的癌症之一。在台灣也是國人十大癌症之一，而在男性癌症死亡率更是排名第四的癌症。神經纖毛蛋白質-1(Neuropilin-1; NRP1)已知在較惡性以及高轉移性的腫瘤中會較高量表現。訊息傳遞轉錄活化基因-3 (Signal transducers and activator of transcription 3; Stat3) 是致癌性轉錄因子，已知在癌細胞和腫瘤微環境細胞都呈現持續活化的現象，在過去的研究已被證實，可能會去影響腫瘤細胞的轉移，以及腫瘤細胞的增生和凋亡。在本研究中，我們想要去探討在口腔癌症中NRP1與Stat3扮演怎樣的角色。首先我們利用免疫組織化學染色(immunohistochemistry)分析OSCC臨床檢體，發現NRP1、Stat3及pStat3的表現量在腫瘤組織中都高於正常組織，而且在腫瘤組織位置上有正相關高表現的現象。另外我們也利用西方墨點法(Western blot)分析四種OSCC細胞株，也發現NRP1及pStat3在較惡性的OSCC細胞株表現量較高。此外我們將免疫組織化學染色的結果進行量化及分析，發現在OSCC中NRP1的表現與腫瘤的期數有正相關，而Stat3則無差異。分析NRP1對OSCC的影響，我們發現NRP1 knockdown時，細胞的移行(migration)，侵入(invasion)及增生(proliferation)都會受到抑制。另外我們發現當細胞knockdown Stat3情況下，NRP1及IL-6的表現量都有明顯的下降，我們利用線上軟體預測在NRP1的promoter上可能具有Stat3 DNA binding site，接著利用luciferase report assay分析，發現在有送入c-Stat3的組別中螢光素酶的活性相較於對照組有明顯的上升，也就表示在口腔癌細胞株中Stat3可能結合在NRP1驅動子上的DNA結合位，進而調控NRP1基因的轉錄表現。在動物實驗方面，我們將knockdown NRP1（shNRP1），Stat3（shStat3）或shLuci的SCC15細胞株分別進行腫瘤異體移植實驗，結果顯示shNRP1及shStat3的組別可以有效的降低腫瘤的大小及腫瘤重量，其中又以shNRP1的組別效果更為顯著。將腫瘤取下進行染色分析,發現在shNRP1及shStat3組別中腫瘤的CD31及Ki67染色有明顯低於shLuci組的表現。總結以上結果，NRP1的表現及Stat3的活化在口腔癌中扮演重要角色，且NRP1與Stat3的相互調控在口腔癌是新的發現，未來可能可以做為口腔癌診斷及治療標靶之參考。

Oral squamous cell carcinoma (OSCC) is a common neoplasm worldwide. In Taiwan, that is also one of the top ten malignancies, and ranked the fourth leading cause of cancer death in Taiwanese male. Neuropilin-1 (NRP1) has been known to be highly expressed in high grade and metastatic tumors. Signal transducers and activator of transcription 3 (Stat3) is a cytoplasmic transcription factor which is constitutively activated in cancer cells and tumor microenvironment and implicates tumor metastasis, proliferation and apoptosis. Our preliminary results showed that Stat3 activation may positively regulate NRP1 expression in lung cancer cells. In this study, we aim to figure out the role of NRP1 and Stat3 in oral cancer. Above all, we used immunohistochemistry to examine the clinical OSCC specimens and found that NRP1/Stat3/p-Stat3 were higher expression in tumors compared to normal tissues and had positive correlation in tumor tissue. The expression level of NRP1 is positively correlated with tumor stage in clinical OSCC specimens. Moreover, the expression of NRP1 and p-Stat3 was higher and positively correlated in highly malignant OSCC cell lines. To investigate the influence of NRP1 in OSCC, we knocked down the expression of NRP1 and found the cell migration, invasion, and proliferation was all decreased in NRP1 silencing cells. In addition, we found that silencing the expression of Stat3 leads to a significant reduction in the expression level of NRP1 and IL-6. We analyzed the promoter region of NRP1 and found a potential Stat3 binding site may involve in transcriptional regulation of NRP1. Next, we use luciferase report assays to verify the regulation of stat3 in NRP1 expression, and then we finding that Stat3 could regulate NRP1 expression by drive on it’s promoter region. In animal models, we established tumor xenografts by subcutaneously implanting SCC15 cells with knockdown of NRP1, stat3 and shLuci (control) respectively. The result show that knockdown of NRP1 or Stat3 could effectively reduce tumor volume and tumor weight, especially in the shNRP1 group. However, analysis for angiogenesis and proliferative effect in the outcome. Tumors from the shNRP1 and shStat3 groups showed significantly lower CD31 and Ki67 staining than the shLuci groups. According to these results, the correlation of NRP1 expression levels and Stat3 activation is a novel finding in oral cancer development. In the future, NRP1/stat3 axis can be a new prognosis marker and therapeutic target in oral cancer.

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