Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49868
Title: | 找尋並探討胃腺癌上皮細胞中微小核醣核酸21之可能標的蛋白 To discover and characterize potential miR-21 targets in gastric adenocarcinoma cells |
Authors: | Ting-Yu Fan 范婷羽 |
Advisor: | 周綠蘋(Lu-Ping Chow) |
Keyword: | 胃癌,幽門螺旋桿菌,微小核醣核酸21,ASPP2, Gastric cancer,Helicobacter pylori,miR-21,ASPP2, |
Publication Year : | 2016 |
Degree: | 碩士 |
Abstract: | 胃癌是全球十大癌症死因之一,在開發中國家較為常見。誘發胃癌產生的影響因子包含了宿主本身的免疫反應、生活環境與飲食習慣等,其中幽門螺旋桿菌 (Helicobacter pylori) 感染為最主要的原因。過去已有研究顯示幽門螺旋桿菌的感染確實與胃癌發生有關,然而其詳細的致癌機轉仍不清楚。
微小核醣核酸 (miRNAs) 為具有約22個核甘酸的小分子RNA,能藉由與特定mRNA基因的3端不轉譯區 (3’UTR) 結合,抑制mRNA轉譯或造成其裂解,進而調控相關的基因表現。近年來已有報導指出幽門螺旋桿菌感染後,會使細胞內miRNAs有異常的表現,其中miR-21為已知的致癌miRNA (oncomir),在許多癌症中都有大量表現的情形,並可藉由抑制其下游基因表現導致癌症產生,因此探討miR-21調控的下游目標基因有助於了解胃癌的發生。 為了釐清miR-21是藉由調控哪些目標基因影響胃腺癌上皮細胞AGS的功能,本實驗室利用了SILAC定量質譜學方法與預測miRNAs目標基因的資料庫miRsystem找到了6個miR-21的潛力目標,分別為DAXX、MAP3K1、NFAT5、PDCD4、RASA1及ASPP2。這6個潛力目標中,我們發現ASPP2 mRNA表現量在miR-21大量表現的細胞中下降情形最為顯著,並且也觀察到大量表現miR-21會減少ASPP2蛋白表現,因此讓我們對ASPP2產生了興趣。我們利用報導基因分析 (luciferase report assay) 證實ASPP2的確為miR-21的目標基因,同時也利用慢病毒攜帶shRNA方法抑制AGS細胞中ASPP2的表現,觀察其參與之功能。實驗結果發現抑制ASPP2表現量會增強細胞增生能力與抑制細胞凋亡,並且也會增加細胞貼附性與非貼附性群落形成的能力。進一步我們利用免疫組織化學染色觀察到人類胃癌組織中ASPP2的表現量低於在胃炎組織的表現。因此我們提出一結論,高度表現的miR-21會抑制其下游目標基因ASPP2的表現,進而導致胃癌的發生。 Gastric cancer (GC) is one of the leading causes of cancer death in the world and more common in developing countries. The interaction of host, bacteria, and environmental factors involve in the development of gastric cancer. Among these, Helicobacter pylori infection is the most common risk factor of GC. Although the role of H. pylori in gastroduodenal diseases has been proposed, the detailed molecular pathway remains unclear. MicroRNAs (miRNAs) are small approximately 22-nucleotide RNAs that modulate gene expression via binding to the 3’ untranslated region (3’UTR) of target mRNAs resulting in translational repression or degradation. Recent studies have suggested that some miRNAs expression is dysregulated in human gastric cells by H. pylori infection. Among these, miR-21 is a well-known oncomir which is upregulated in many cancers and leads to tumorigenesis by regulating its downstream target genes. Therefore, investigating downstream target genes of miR-21 could help us to understand the incidence of gastric cancer. In order to clarify how miR-21 affects the function of AGS cells, we used SILAC-based quantitative MS and miRsystem, an integrated system for predicting of miRNA targets, to find the potential target genes of miR-21. Finally, we found 6 potential targets, including DAXX, MAP3K1, NFAT5, PDCD4, RASA1, and ASPP2. Among 6 potential targets, we observed that the expression of ASPP2 mRNA was decreased most significantly in miR-21-overexpressed cells. Overexpression of miR-21 also decreased the expression of ASPP2 protein. Then we also found that miR-21 could bind to the 3’UTR of ASPP2, suggesting that ASPP2 is a direct target of miR-21. In order to realize the function of ASPP2, we knockdown of ASPP2 in AGS by using lentivirus-mediated shRNA. The results showed that knockdown of ASPP2 increased cell proliferation and decreased 5-Fu-induced apoptosis in AGS. Knockdown of ASPP2 also increased anchor-dependent and anchor-independent colony-forming ability of AGS cells. Furthermore, we found that the expression of ASSP2 was lower in human gastric cancer tissue than in gastritis tissue. As a result, we concluded that ASPP2 is down-regulated by miR-21 overexpression and may lead to gastric carcinogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49868 |
DOI: | 10.6342/NTU201602338 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-105-1.pdf Restricted Access | 3.56 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.