博碩士論文 992213011 詳細資訊



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姓名 陳彥如(Yen-Ju Chen)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 利用微核糖核酸及全基因體定序分析法 探討牛樟芝對肝癌細胞的作用機制
(Global assessment of Antrodia cinnamomea crude extract -induced microRNA and transcriptomic alterations in hepatocarcinoma cells)
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摘要(中) 牛樟芝(學名 Antrodia cinnamomea或 Antrodia Camphorata)為台灣特有種，長久以來被用於治療多種疾病，包括腹瀉、胃痛、食物中毒、高血壓，以及癌症(包含肝癌)。經Annexin V、caspas 3&7染色以及DNA片段化實驗，我們發現牛樟芝子實體酒精萃取物，能在兩小時內有效抑制SK-HEP-1人類肝癌細胞生長，促使肝癌細胞進行凋亡。以往樟芝的研究著重於長時間的特定基因試驗與動物模式，對其抗癌機制仍未完全了解。我們利用客觀且全面性的次世代定序法，分析微核糖核酸(microRNA)以及轉錄體的表達，全面性研究樟芝的抗癌途徑。由定序結果我們發現樟芝能夠全面性抑制SK-HEP-1肝癌細胞的miRNA表達，而不太影響BNL CL.2正常老鼠肝細胞。
由實驗結果我們推測牛樟芝早期的抗癌機制，可能是藉由調控miRNA代謝的蛋白酶Dicer、Drosha以及XRN2，而造成miRNA表達量下降，進一步影響標的基因的訊息傳遞。將轉錄體資料進行GO term及訊息傳遞路徑分析，顯示樟芝能活化癌症凋亡機制並且抑制MAPK/ERK以及PI3K/Akt/NFκB等癌症重要路徑以抑制癌細胞生長。
摘要(英) Antrodia cinnamomea, an endemic fungus of Taiwan, is well known as a herbal medicine to remedy various illnesses, including liver cancer, a major cause of death in Asia. However, the early mechanism of its anti-cancer effect was unclear. In this study, we explored the anti-cancer mechanism induced by Antrodia cinnamomea fruiting body ethanol crude extract (AcFBE) on human hepatocellular carcinoma cell (SK-HEP-1) through in vitro assay in conjunction with miRNA and mRNA transcriptome profiling using next generation sequencing (NGS). Results indicated that 500μg /ml of AcFBE can induce apoptotic cell death on SK-HEP-1 within 2 hours, as characterized by cell viability assay, cell morphology alteration, cell membrane extroversion, caspases activation, and DNA fragmentation. The sequence data revealed 354 known miRNAs from miRNA libraries. To our surprise, 85.4% of known miRNAs in SK-KEP-1 were decreased, while only 57.3% of known miRNAs were found decreased for normal mouse liver cells (BNL CL.2). The AcFBE-affected miRNAs expression profiles showed dramatic down-regulation in SK-HEP-1 cells, with 95.2% (141/148) and 84.1% (122/145) affected after 2 hr and 4 hr AcFBE treatment, respectively, suggesting that AcFBE caused a global inhibition of miRNA level. This result was supported by western blotting showing decreases of miRNA biogenesis proteins, Dicer and Drosha, together with an increase of XRN2 involved in miRNA degradation. Transcriptome analysis showed 2077 (58.3%) and 1491 (40.1%) up-regulated genes in 2-hr and 4-hr libraries, respectively, based on 2 fold cutoff. We then analyzed 365 genes most significantly regulated by AcFBE using GO term analysis on transcriptome data, indicating a strong association with apoptosis. Overall, our study suggests that AcFBE is able to globally reduce SK-HEP-1 miRNA expression and thereafter affect gene expression regulation, causing cancer cell death by apoptosis. This is the first report implicating miRNA in the anti-cancer effects of A. cinnamomea fruiting body.
關鍵字(中) ★ 抗癌
★ 微核糖核酸
★ 子實體
★ 樟芝		 關鍵字(英) ★ anti-cancer
★ microRNA
★ fruiting body
★ Antrodia cinnamomea
論文目次 Chinese Abstract i
English Abstract ii
Table of Contents iii
List of figures v
List of tables vi
Abbreviation list vi
Chapter1. Introduction 1
1-1 Antrodia cinnamomea 1
1-2 MicroRNA 2
1-2-1 MicroRNA biogenesis and function 2
1-2-2 MicroRNA in cancer 3
1-3 Apoptosis pathway 3
Chapter 2. Materials and Methods 5
2-1 Cell Cultures 5
2-2 Isolation and maintenance of Antrodia cinnamomea 5
2-3 Preparation of crude extract from the fruiting body and mycelia of A. cinnamomea 6
2-4 Ingredients analysis of AcFBE by HPLC 7
2-5 Experimental flowchart 7
2-6 Pre-sequencing in vitro studies 8
2-6-1 Detect cell viability by MTT assay 8
2-6-2 Detection of apoptosis by Annexin V and PI staining 8
2-6-3 Detection of apoptosis by caspase-3 & 7 assay 9
2-6-4 Detection of apoptosis by DNA fragmentation 9
2-7 Western blotting 9
2-8 Sequencing libraries preparation and construction 10
2-8-1 Experimental design 10
2-8-2 Isolation of total RNA 11
2-8-3 microRNA library construction 11
2-8-4 Fragment library construction 11
2-9 Data processing and bioinformatics analysis 12
2-9-1 Small RNA pipeline and bioinformatics analysis 12
2-9-2 Transcriptome sequence processing and bioinformatics analysis 13
Chapter 3. Results and discussion 14
3-1 Analysis of AcFBE by HPLC 14
3-2 Effect of AcFBE on cell proliferation inhibition was detected by MTT assay 14
3-3 AcFBE resulted in SK-HEP-1 cell death through apoptosis 16
3-4 microRNA sequence processing and bioinformatics analysis 18
3-5 Cross-library comparison of miRNA expression levels 19
3-6 Global down-regulation of miRNA in AcFBE treated SK-HEP-1 cancer cells 20
3-7 miRNA down-regulation is caused by a blind shooting mechanism 22
3-8 Western blot analysis of proteins involved in miRNA metabolism 24
3-9 Transcriptome data processing and bioinformatics analysis 25
3-10 Analysis of transcriptomic alterations caused by AcFBE treatment 26
3-11 Gene Ontology analyses for transcriptome libraries 27
3-12 Pathway analyses for AcFBE-affected genes 28
Chapter 4. Conclusion 31
Chapter 5. References 32
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指導教授 邱國平(Kuo-Ping Chiu) 審核日期 2012-7-4
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