口腔鱗狀細胞癌 (OSCC) 一種局部侵襲性腫瘤，佔頭頸癌的 90%，是全球第 6 位最常見的惡性腫瘤，迄今為止，尚無有效的方法來減少 OSCC 患者的腫瘤惡性發展。 MicroRNAs (miRNAs) 是蛋白質編碼基因的有效轉錄後調節因子。癌症中 miRNA 的失調意謂著 miRNA 在腫瘤發生中可能扮演關鍵角色。近期有許多文獻報導證實，外泌體為miRNA的重要載體，在腫瘤微環境的建立中扮演重要角色。因此，我們假設源自 OSCC 的外泌體含有致癌 miRNA。使用 miRNA 微陣列結合 OSCC 患者的 miRNA 表達圖譜(GSE45238)，我們發現與正常角質形成細胞衍生的外泌體相比，miR-421在 OSCC 細胞衍生的外泌體中是高度表達。在一系列實驗中我們證實miR-421 能夠誘導血管新生並增加血管內皮細胞中VEGFR2、ERK、Akt磷酸化的表現。此外，miR-421能夠藉由抑制硫酸乙酰肝素 2-O-磺基轉移酶 (HS2ST1)蛋白的表現來誘導血管新生反應，HS2ST1 是一種介導硫酸乙酰肝素蛋白聚醣2-O-硫酸化的酶。總之，這些數據表明 miR-421 在調節HS2ST1/VEGFR2/ERK 或 /Akt 在血管新生中有著重要角色。因此，抑制 miR-421 表達或阻斷這些外泌體的傳輸可能是 OSCC 的一種新的抗血管新生治療策略。

Oral squamous cell carcinoma (OSCC), a characteristic locally aggressive tumor, is the 6th most common malignancy worldwide OSCC accounting for about 90% of head and neck cancer. Thus far, no effective methods have been available to reduce the malignant progression in OSCC patients. MicroRNAs (miRNAs) are potent post‐transcriptional regulators of protein coding genes. Patterns of dysregulation of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. Recently, exosomes were established to be significant carriers of miRNAs, which play an important role in the establishment of the tumor microenvironment. Therefore, we hypothesized that exosomes derived from OSCC contain oncogenic miRNAs. Using miRNA microarray combined with OSCC patients’ miRNA expression profile (GSE45238), we identified miR-421 which was highly expressed in OSCC cells-derived exosomes compared with normal keratinocyte-derived exosomes. Series of experiments involving the introduction of exogenous miR-421 induced angiogenic cascade increasing VEGFR2 activity, ERK phosphorylation, Akt phosphorylation and tube formation in human umbilical vein endothelial cells (HUVEC). Furthermore, miR-421 triggered the angiogenic cascade by targeting heparan sulfate 2-O-sulphotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of heparan sulfate proteoglycans. Taken together, these data indicate that miR-421 plays an important role in mediating HS2ST1/VEGFR2/ERK or /Akt angiogenic cascade. In addition, suppressing the miR-421 expression or blocking the transmission of these exosomes might be a novel antiangiogenic therapeutic strategy for OSCC.

摘要 i
Abstract ii
致謝 iii
1. Introduction 1
1.1 Oral squamous cell carcinoma (OSCC) 1
1.2 Exosomes 1
1.3 MicroRNA 2
1.4 Angiogenesis 3
1.5 HS2ST1 4
1.6 Specific aims: 4
2. Materials and Methods 6
2.1 Exosome purification 6
2.2 RNA extraction and Reverse-Transcriptase PCR (RT-PCR) 6
2.3 Cell lines and culture condition 6
2.4 Transfection experiments 7
2.5 Protein Extraction and Western Blot Analysis 7
2.6 Cell Migration and Invasion Assay 8
2.7 Tube Formation Assay 8
2.8 Transmission electron microscope (TEM) 8
2.9 Virus production and infection of target cells 8
3. Results 10
3.1 Characteristics of oral cancer-derived exosomes. 10
3.2 OSCC-derived exosomes affect HUVEC migration, invasion and tube formation abilities. 10
3.3 Exosomal microRNA affect angiogenesis 11
3.4 MiR-421 affect HUVEC migration, invasion ability. 11
3.5 Exosomal miR-421 affect HUVEC migration, invasion and angiogenesis ability. 11
3.6 HS2ST1 is a direct target of miR-421 in HUVEC. 12
3.7 HS2ST1 level affect HUVEC migration, invasion and angiogenesis ability. 13
.3.8 HS2ST1 level affect VEGFR2, Akt, Erk signaling pathway in HUVEC. 13
3.9 HS2ST1 is able to block miR-421 mediated promotion of migration, invasion and angiogenesis ability in HUVEC. 13
3.10 HS2ST1 is able to block miR-421 mediated affect VEGFR2, AKT, ERK signaling pathway in HUVEC. 14
3.11 HS2ST1 is able to block exosome delivered miR-421 mediated promotion of migration, invasion and angiogenesis ability in HUVEC. 14
3.12 HS2ST1 is able to block OSCC exosome delivered miR-421 mediated effect of AKT, ERK single pathway in HUVEC. 14
Discussion 15
References 34

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