臺灣博碩士論文加值系統

攝護腺癌 (Prostate cancer，PCa) 是全球男性癌症死亡主因之一，發病率與年齡呈正相關，儘管能夠利用雄性激素剝奪療法 (androgen deprivation therapy，ADT) 治癒，仍有許多患者會在數月至數年後復發，發展成去勢抗性攝護腺癌 (CRPC)，在人口高齡化的問題下，越來越多患者在ADT治癒後，足以存活到PCa復發，形成CRPC。CRPC除了對ADT有抗性，也具高度轉移及化療抗性，主要特徵是重新活化雄性賀爾蒙受體 (Androgen receptor) 的訊息傳遞 (AR signaling)，然而還有一群不表現AR的PCa細胞，會表現神經內分泌樣分化 (NED) 標誌，被提出為發展成CRPC的新特徵，稱為NEPC (Neuroendocrine prostate cancer)。近幾年研究發現許多NED誘導因子被發現，如ADT、IL-6與缺氧 (hypoxia)，其中調節NED路徑的分子REST (repressor element-1 silencing transcription factor) 被發現在NED的PCa中表現顯著降低，同時發現在IL-6或是低氧處理的PCa其細胞自噬 (autophagy) 機制會被活化。
全基因體表達分析顯示出長鏈非編碼RNA (lncRNAs) 能夠做為癌症新標誌，在剔低REST表現的全基因體分析，利用NONCODE v4資料庫分析，找到可能參與NED的lncRNA，有潛力成為鑑定NEPC的新生物標誌，並進行小規模之siRNA篩選，鑑定出LINC01801在REST剔低後表現量上升，對於NED是必須的，因此將針對LINC01801研究其在PCa轉變為NEPC的重要性。
本研究發現，LINC01801參與攝護腺癌NED的過程，且一旦敲低LINC01801的表現，其NED現象也會受到抑制，在RNA-Seq結果也得知，LINC01801也參與細胞自噬的活化，而在miR-Seq結果篩選出has-miR-6889-3p可能與LINC01801有交互作用，同時也受LINC01801所調控，在預測標靶基因也觀察到has-miR-6889-3p可能與細胞自噬相關基因有交互作用，故我們推測LINC01801可能藉由調控has-miR-6889-3p而活化細胞自噬的機制。

　　Prostate cancer (PCa) is one of the leading cause of cancer death in men worldwide. Although PCa can be successfully treated by androgen deprivation therapy (ADT), many patients will relapse to castration-resistant prostate cancer (CRPC) months to years after treatment. Due to the incidence of PCa is exponentially correlated with age and the global aging, more and more patients relief from PCa by ADT treatment are now live long enough to develop CRPC. CRPC, is an advanced type of PCa that requires immediate attention since it is not only no longer responds to ADT but also highly chemoresistant and metastatic. Although reactivation of AR signaling is a major feature of CRPC, AR-null tumors express neuroendocrine differentiation (NED) markers has also been proposed as a new feature, so called NEPC (Neuroendocrine prostate cancer). In recent years, many NE differentiation (NED) inducers, such as ADT, IL-6 and hypoxia, were identified. Also find out that repressor element-1 silencing transcription factor (REST), a transcription factor that regulates NED pathways, was significantly reduced in NEPC. Moreover, our data shows that autophagy mechanism was activated by IL-6 or hypoxia treatment.
Integrative genome-wide expression analysis revealed long non-coding RNAs (lncRNAs) are emerging as novel determinants and prognostic markers of various cancers, including PCa. To discover novel NED-associated lncRNAs, we reannotated our previously published RNA-seq data obtained from REST knockdown PCa LNCaP cells using NONCODE v4 database. Following a small scale siRNA knockdown screening covering the identified NED-associated lncRNAs and RT-qPCR verification, LINC01801 were identified to be essential for NED. Therefore, we focus on LINC01801 and study its importance of NED.
In this study, we found that LINC01801 is essential for NED knockdown. Pathway analysis of RNA-Seq, the results show that LINC01801 is involved in the activation of autophagy. We also performed the Small RNA-Seq. The results show that has-miR-6889-3p is down-regulated by LINC01801.The results suggest that hsa-miR-6889-3p is a downstream target of LINC01801. By using the target gene prediction, there were some autophagy related genes which may be regulated by has-miR-6889-3p. Therefore, we hypothesis that, LINC01801-has-miR-6889-3p aixs potential activate autophagy in NED cells.

目錄
誌謝 i
中文摘要 ii
Abstract iii
目錄 v
一、緒論 1
1. 攝護腺癌 (Prostate Cancer) 1
1.1 攝護腺癌 1
1.2 攝護腺癌的診斷 1
1.3 攝護腺癌的治療 2
1.4 攝護腺癌的復發 2
2. 神經內分泌分化 (Neuroendocrine differetiatiton，NED) 3
2.1 攝護腺癌的神經內分泌分化 3
2.2 神經內分泌分化的誘導因子 3
2.3 神經內分泌攝護腺癌與神經內分泌分化 4
3. REST (RE1-silencing transcription factor) 4
3.1 REST 4
3.2 REST與神經內分泌分化 5
4. 長鏈非譯碼RNA (Long non-coding RNA，lncRNA) 5
5. 細胞自噬 (Autophagy) 6
二、研究目的 8
三、實驗材料 9
1. 菌株 9
2. 質體 9
3. 細胞株 9
4. 培養液 9
5. 一般溶液 10
6. 抗體 11
7. 引子 11
8. 攝護腺癌臨床檢體 11
四、實驗方法 12
1. 細胞培養 12
2. RNA 次世代高通量定序 (RNA-seq) 及gene ontology (GO) 分析 12
3. 小片段RNA定序 (Small RNA Sequencing, smRNA-Seq)分析 12
4. RNA製備 12
5. 反轉錄作用 (Reverse transcription) 13
6. 即時聚合酶連鎖反應 (RT-qPCR) 13
7. SDS-聚丙烯醯胺凝膠電泳 13
8. 西方墨點法 (Western blot) 14
9. 組織免疫染色 (IHC-staining) 14
10.細胞活性 (MTT assay 15
11. 細胞轉染 (Transfection) 15
12. 轉型作用 (Transformation) 15
13. 細胞轉染 (Transfection) 15
14. 感染 (Infection) 16
15. Neurite outgrowth染色及顯微鏡定量 Neurite Outgrowth Staining Kit 16
16. 免疫螢光染色 (Immunofluorescence assay，IFA) 16
17. TCGA 分析 (The Cancer Genome Atlas) 17
18. miRNA的目標基因預測 17
19. Statistical Analysis 17
五、實驗結果 18
1. 利用次世代定序分析並找尋在REST剔低誘導NED的PCa受REST所調控lincRNA 18
2.確認LINC01801 與神經內分泌分化之關聯 18
3.探討LINC01801在NED的機轉 19
4. LINC01801對autophagy機制之影響 20
5. LINC01801與microRNA 21
6. LINC01801對於腫瘤進展之影響 22
六、討論 24
1, LINC01801對於細胞走向NED的影響 24
2. 細胞自噬對於攝護腺癌的影響 24
3. LINC01801與hsa-miR-6889-3p 25
3. LINC01801在臨床上的意義 25
七、參考文獻 26
八、圖與表 31

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