利用核酸定序技術研究麩醯胺酸匱乏對大腸癌細胞外泌體RNA組成的影響

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劉柏渝(Bo-Yu Liu) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

利用核酸定序技術研究麩醯胺酸匱乏對大腸癌細胞外泌體RNA組成的影響
(Exploring the Impact of Glutamine Deprivation on RNA Cargo Profiles of Colorectal Cancer Cell Exosomes by Nucleotide Sequencing)

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★ 定義新型前列腺癌致癌及轉移驅動因素

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至系統瀏覽論文 (2029-9-20以後開放)

摘要(中)

麩醯胺酸（Glutamine）在癌細胞中扮演著重要的角色。作為一種非必需胺基酸，其在正常細胞中的需求遠低於腫瘤細胞。在癌症細胞中，麩醯胺酸主要通過麩醯胺酸分解代謝（glutaminolysis）途徑被代謝，轉化為麩胺酸（glutamate）和氨（ammonia），進一步生成α-酮戊二酸（α-Ketoglutarate）並進入三羧酸循環（TCA cycle），從而為腫瘤細胞的快速生長和增殖提供能量和代謝中間體。在腫瘤微環境中，由於癌細胞的大量消耗，麩醯胺酸往往呈現匱乏狀態。這種匱乏狀態對外泌體的產生和功能有顯著影響。有研究表明，麩醯胺酸匱乏會增加生成於Rab11陽性回收內涵體(recycling endosomes)的癌細胞外泌體數量，這些外泌體中攜帶的分子能夠影響腫瘤微環境中的其他細胞，幫助腫瘤適應代謝壓力。外泌體是由細胞釋放的微小細胞外囊泡，其直徑在30~150 nm，其中富含蛋白質、核酸和脂質。其中，RNA分子在外泌體中的功能極為關鍵，它們能夠被轉移到腫瘤微環境中的其他細胞，執行功能，從而促進腫瘤的增長和擴散。近年來，有研究利用定序技術，如奈米孔定序和NGS Illumina平台，進一步探討外泌體中RNA的組成以及推測其生物機制功能，這些先進的定序技術不僅提供了對外泌體RNA種類和豐富度的全面觀察，還能使研究者能夠識別特定RNA分子在癌症進程中的角色。由於麩醯胺酸匱乏與癌症惡化相關的研究仍然不足，因此，本研究利用奈米孔定序和NGS定序來研究麩醯胺酸匱乏下的外泌體的RNA組成。本研究發現，大腸癌HCT116細胞在正常麩醯胺酸與麩醯胺酸匱乏下分泌的外泌體RNA組成並不相同。重要的是，藉由KEGG訊息路徑富集分析，我們發現這些表現量不同的RNA的蛋白產物集中參與於RAS、PI3k-AKT與Rap1訊息傳遞，而這些訊息傳遞路徑多與癌症惡化有關。因此，本研究增進了我們對於麩醯胺酸匱乏所誘發之外泌體的功能了解。我們期待這些研究發現不僅能夠增進對腫瘤生物學的理解，也為發展新的癌症治療策略提供了可能。

摘要(英)

Glutamine plays a crucial role in cancer cells. As a nonessential amino acid, its demand in tumor cells far exceeds that in normal cells. In cancer cells, glutamine is primarily metabolized through the glutaminolysis pathway, converting into glutamate and ammonia, further synthesizing α-ketoglutarate and entering the tricarboxylic acid (TCA) cycle, thereby providing energy and metabolic intermediates necessary for the rapid growth and proliferation of tumor cells. In the tumor microenvironment, due to the extensive consumption by cancer cells, glutamine often becomes depleted. This state of depletion significantly affects the production and function of exosomes. Studies have shown that glutamine depletion can increase the number of cancer exosomes released by Rab11-positive recycling endosomes, which carry molecules that can affect other cells in the tumor microenvironment and help the tumor adapt to metabolic stress. Exosomes are tiny extracellular vesicles released by cells, ranging in size from 30 to 150 nm and rich in proteins, nucleic acids, and lipids. Particularly, the role of RNA molecules within exosomes is crucial; they can be transferred to other cells in the tumor microenvironment, thus promoting tumor growth and disease spread. Recent studies using sequencing technologies, such as the nanopore sequencing and the NGS Illumina platforms, have further explored the RNA composition, leading to a better understanding of biological mechanisms of exosomes. These advanced sequencing techniques not only provide a comprehensive observation of the types and abundance of RNA within exosomes but also enable researchers to identify specific RNA molecules′ roles in cancer progression. Due to the insufficient research on the relationship between glutamine deprivation and cancer progression, this study utilizes nanopore sequencing and NGS sequencing to investigate the RNA composition of exosomes under glutamine deprivation. This study found that the RNA composition of exosomes secreted by colorectal cancer HCT116 cells differs between normal glutamine conditions and glutamine deprivation. Importantly, using the KEGG pathway enrichment analysis we found that the protein products of these differentially expressed RNAs are primarily involved in the RAS, PI3K-AKT, and Rap1 signaling pathways, which are often associated with cancer progression. Therefore, this study enhances our understanding of the functions of exosomes induced by glutamine deprivation. We hope that these findings will not only advance the understanding of tumor biology but also provide potential avenues for developing new cancer treatment strategies.

關鍵字(中)

★ 外泌體
★ 大腸癌細胞
★ 核酸定序

關鍵字(英)

★ Exosomes
★ Colorectal Cancer Cell
★ Nucleotide Sequencing

論文目次

目錄
中文摘要 i
Abstract iii
致謝 v
目錄 vi
圖目錄 x
表目錄 xi
中英文對照表 xii
第一章緒論 1
1.1 外泌體簡介以及重要性 1
1.1.1外泌體功能簡介 2
1.1.2外泌體生成過程 3
1.1.3 外泌體攜帶各式分子-蛋白質、脂質及代謝物 5
1.1.4外泌體攜帶各式分子-核糖核酸 7
1.1.5 外泌體攜帶作為疾病分子標記 8
1.1.6 RNA分子如何運送至外泌體 9
1.2 腫瘤微環境介紹 10
1.2.1 癌細胞中的麩醯胺酸代謝 11
1.2.2 癌細胞對麩醯胺酸匱乏的反應 13
1.2.3 腫瘤微環境對外泌體的影響 14
1.3 大腸癌簡介 15
1.3.1 大腸癌惡化生物機制 16
1.3.2 外泌體中的RNAs如何影響大腸癌 17
1.4 外泌體所攜帶RNA分子之基因定序分析研究 19
1.4.1 NGS技術原理 19
1.4.2 Nanopore技術原理 21
1.4.3 NGS跟Nanopore定序技術比較 23
1.5 研究動機 24
第二章實驗方法與材料 25
2.1實驗方法 25
2.1.1外泌體研究中的HCT116大腸癌細胞株培養與處理 25
2.1.2麩醯胺酸剝奪條件下的細胞外泌體分離 25
2.1.3分離外泌體 26
2.1.4西方墨點法 27
2.1.5抽取外泌體RNA 28
2.1.6使用Kapa hyper kit (Oxford Nanopore Technology)建庫 29
2.1.7 RNAseq 資料分析 31
2.2 實驗材料 32
第三章結果 33
3.1 SEC分離後外泌體標記物的西方墨點法分析 33
3.2以西方墨點法確認麩醯胺酸匱乏造成外泌體的蛋白質組成改變 34
3.3 RNA濃度測定方法比較與應用 35
3.4 使用奈米孔定序分析HCT116細胞外泌體RNA 37
3.4.1 利用ONT定序數據分析分布於Gln+ exosomes與Gln- exosomes中的RNA 38
3.4.2 外泌體中RNA類型分析 39
3.4.3 Gln+ exosomes和Gln- exosomes 中RNA類型分佈分析 40
3.4.4 ONT定序之Gln- 與 Gln+ 外泌體中不同 RNA 類型的 CPM 及百分比比較 41
3.4.5 HCT116外泌體RNA組成在不同麩醯胺酸濃度培養下表達差異 43
3.5基於ONT定序結果選定 LOC105378179與C8orf34-AS1 RNA進行引物設計及RT-qPCR分析 44
3.5.1 利用IGV基因圖分析LOC10537817基因的轉錄變體與引物設計 44
3.5.2 利用IGV基因圖分析C8orf34-AS1基因的轉錄變體與引物設計 45
3.5.3外泌體RNA內參基因表達水平分析與選擇 46
3.5.4 外泌體中C8orf34-AS1與LOC105378179 表現量分析 47
3.6 利用NGS (next generation sequencing)定序技術分析HCT116細胞外泌體RNA在不同麩醯胺酸條件下的組成 50
3.6.1 利用NGS定序數據分析分布於Gln+ exosomes與Gln- exosomes中的RNA 50
3.6.2 Gln+ Exosomes與Gln-Exosomes樣本中的RNA類型分佈分析 52
3.6.3 NGS定序之Gln- 與 Gln+ 外泌體中不同 RNA 類型的 TPM 及百分比比較 53
3.6.4 NGS定序結果分析麩醯胺酸濃度對HCT116外泌體之RNA組成的影響 55
3.6.5 HCT116 Gln- Exosomes中上調與下調基因類別分析 56
3.6.6 HCT116 Gln- Exosomes中上調與下調基因GO分析 57
3.6.7 HCT116 Gln+ Exosomes與Gln- Exosomes樣本中差異表達基因的GO功能網絡分析 58
3.6.8 HCT116細胞外泌體在麩醯胺酸供應變化下差異表達基因的KEGG訊息路徑富集分析 59
3.7 NGS發現之差異表現基因與ONT發現之基因的重疊分析 61
第四章.討論 63
4.1 麩醯胺酸匱乏培養下造成HCT116癌細胞分外泌體RNA組成的改變 63
4.2 麩醯胺酸匱乏對HCT116細胞外泌體RNA組成的影響分析 64
4.3 由基因差別表現量分析推測HCT116細胞在麩醯胺酸匱乏壓力下所分泌外泌體的功能 65
4.4 外泌體RNA定量分析之內參基因選擇 66
4.5 LOC105378179 RNA在Gln- exosomes中顯著增加 67
4.6 未來研究方向 68
參考文獻 69

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指導教授

范世榮(Shih-Jung Fan)

審核日期

2024-9-20

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