臺灣博碩士論文加值系統

癌細胞轉移是影響卵巢癌患者生存的關鍵因素，然而目前對於卵巢癌轉移的分子作用機制仍不清楚。本研究比較同源性之高低侵入性卵巢癌細胞株，發現微型核糖核酸-138及708在高侵入性細胞中，均呈現表現量下降的現象，此二者微型核糖核酸均能夠抑制癌細胞的侵入能力。利用異種原位癌症模型，我們證實微型核糖核酸-138具有抑制小鼠卵巢癌轉移至其它器官的功能。此外，SOX4及HIF-1α是微型核糖核酸-138的直接目標基因，且微型核糖核酸-138所抑制的卵巢癌細胞侵入現象，能為SOX4及HIF-1α的大量表現而回復。尋找下游訊息傳遞路徑的過程中，發現SOX4能夠直接影響轉錄EGFR；而 HIF-1α 則透過蛋白質降解的路徑來調控Slug。於臨床檢體分析方面，發現卵巢癌晚期之臨床檢體呈現微型核糖核酸-138表現量低；SOX4表現量高的現象。 檢體具有微型核糖核酸-138低表現且SOX4高表現型態的患者，則與大量腹水累積和癌細胞淋巴結轉移等惡性腫瘤臨床現象呈現高度相關性。另一具有抑制卵巢癌細胞侵入能力的微型核糖核酸-708，研究證實它能直接調控Rap1B的表現量。而糖皮質激素受體-α可能是轉錄微型核糖核酸-708表現的重要調控因子，由細胞實驗中亦可證實dexamethasone具有刺激微型核糖核酸-708表現，進而抑制卵巢癌細胞侵入能力的作用。從臨床檢體的分析中，發現與正常組織相較之下，卵巢癌檢體呈現微型核酸核酸708低表達；Rap1B高表達的趨勢，印證體外細胞實驗的結果。總括，本研究探討微型核糖核酸-138及708於卵巢癌轉移的功能角色及臨床關聯性，藉此提供具有應用潛力的分子診斷生物標記分子及未來治療卵巢癌轉移的方針。

Metastasis is the major factor affecting patient survival in ovarian cancer. However, its molecular mechanisms remain unclear. The present study used isogenic pairs of low and high invasive ovarian cancer cell lines to demonstrate the downregulation of miRNA-138 and miRNA-708 in the highly invasive cells, and their functioning as an inhibitor of cell migration and invasion. An orthotopic xenograft mouse model further demonstrated that the expression of miRNA-138 inhibited ovarian cancer metastasis to other organs. Results indicated that miRNA-138 directly targeted SOX4 and HIF-1α, and overexpression of SOX4 and HIF-1α effectively reversed the miRNA-138-mediated suppression of cell invasion. Epidermal growth factor receptor (EGFR) acted as the downstream molecule of SOX4 by way of direct transcriptional control, whereas Slug was the downstream molecule of HIF-1α by way of proteasome-mediated degradation. Analysis of human ovarian tumors further revealed downregulation of miRNA-138 and upregulation of SOX4 in late stage tumors. Patients with miRNA-138low/ SOX4high signature are predominant in late stage and tend to have malignant phenotypes including lymph nodes metastasis, larger ascites volume and higher tumor grade. For miR-708, we found it suppressed ovarian cancer cells invasion and directly targets Rap1B. Glucorcoticoid receptor-alpha, a transcriptional factor activated by dexamethasone, might be the upstream regulator of miRNA-708. Our cell-based study revealed a novel role of miRNA-708 in the inhibition of ovarian cancer cell invasion by dexamethasone. Clinically, expression of miRNA-708 was downregulated while that of Rap1B was upregulated in malignant human ovarian cancers compared to normal tissues. This study demonstrates the functional role and clinical relevance of miRNA-138 and miRNA-708 in ovarian cancer cell invasion and metastasis, providing a potential therapeutic strategy and diagnosis markers for ovarian cancer.

Abstract 1
摘要 3
誌謝 4
Table of contents 6
List of figures 9
List of tables 11

Chapter 1: Introduction and literature review 12
1.1 Clinical overview of ovarian cancer metastasis 12
1.2 Current knowledge about the molecular mechanisms of ovarian cancer metastasis 13
1.3 MicroRNAs 14
1.4 Specific aim and impact of this study 15

Chapter 2: Methodology 17
2.1 Stragety and rationale 17
2.2 Cell culture 17
2.3 Human ovarian tumor samples 17
2.4 Cell adhesion assay 18
2.5 Paclitaxel resistance assay 18
2.6 Soft agar colony formation assay 18
2.7 Cell migration, invasion assay and in vitro selection 19
2.8 Tail vein metastasis assay and in vivo metastasis selection 20
2.9 Plasmid constructs 20
2.10 Transfection and stable cell line selection 20
2.11 Orthotopic xenograft mouse model 21
2.12 Quantitative real-time PCR for genes and microRNAs 22
2.13 Western blot analysis 22
2.14 3’ UTR luciferase reporter assay 23
2.15 Chromatin immunoprecipitation (ChIP) assay 23
2.16 Fluorescence in situ hybridization, immunofluorescence, and quantification 24
2.17 Statistical analysis 24
2.18 Cell identification 25

Chapter 3: Results 26
3.1 The in vitro-selected invasion ovarian cancer cells exhibit an aggressive cancer cell phenotype 26
3.2 MicroRNA-138 is downregulated in invasive cells and is a negative regulator of ovarian cancer cell invasion and metastasis 27
3.3 MicroRNA-138 suppressed ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1α 28
3.4 Epithelial growth factor receptor and Slug are the downstream mediators of SOX4 and HIF-1α respectively 30
3.5 MicroRNA-138low / SOX4 high signature is a potential prognosis marker for ovarian cancer 32
3.6 MicroRNA-708, downregulated in highly invasive ovarian cancer cells, suppresses cancer cell migration, invasion, and Rap1B expression 33
3.7 Dexamethasone, a glucocorticoid receptor-α agonist, induces miR-708 expression in ovarian cancer cells 34
3.8 MicroRNA-708 is downregulated, and Rap1B is upregulated in malignant human ovarian cancer samples 36

Chapter 4: Discussion and future perspective 38
4.1 Potential upstream regulator of miRNA-138 38
4.2 Role of SOX4 in ovarian cancer 38
4.3 Hypoxia, EMT, and ovarian cancer metastasis 39
4.4 Interaction between SOX4 and HIF-1α 41
4.5 Upstream regulator network of miRNA-708 41
4.6 Role of Dex in cancer treatment 42
4.7 Conclusion and future perspective 43

Reference 46
Tables 51
Figures 62

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