利用蛋白體學技術鑑定受幽門螺旋桿菌感染之人類胃腺癌上皮細胞中微核醣核酸21之標的

Proteomics Approach to Identify miR-21 Targets in Helicobacter pylori-infected AGS Cells

廖瑩竹(Ying-Chu Liao)

指導教授 ： 周綠蘋

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.03138

摘要

無資料

關鍵字

胃癌 ； 幽門螺旋桿菌 ； 微核醣核酸 ； 微核醣核酸21 ； PDCD4

並列摘要

Gastric cancer is the most common malignancy of the gastrointestinal tract in East Asian populations. Although the pathogenic factors involved in gastric carcinogenesis are thought to be multifactorial, infection with Helicobacter pylori (H.pylori) seems to be a main risk factor for the development of gastric cancer nowadays. The significant role of H. pylori on gastrointestinal tract diseases has been proposed, but the detailed mechanism/molecular pathway remains unclear, especially the correlations to miRNAs. MiRNAs are endogenous, small noncoding RNAs, which function by base pairing with the 3’- untranslated region (3’UTR) of target messenger RNAs, thereby regulating as high as 30% of all protein-coding genes expression. One of miRNAs, miR-21, is overexpressed in most tumor types, and acts as an “oncomiR” by targeting many tumor suppressor genes related to numerous cancer-related phenotypes.
To reveal the correlation between H.pylori infection and miR-21, we first verified that miR-21 is overexpressed aberrantly in AGS cells caused by H. pylori infection using qRT-PCR. Following we developed a miR-21 stable cell line which constantly expresses higher level of miR-21 than the mock one. Furthermore, we searched the potential targets of miR-21 using 2-Dimentional Electrophoresis (2DE) to compare the differences proteome between miR-21 stable cell line and mock, following identified the potential target proteins by LC-MS/MS. Continuing analyzed these identified and predicted proteins with IPA (Ingenuity Pathway Analysis) bioinformatics approach, we observed that these potential targets, such as tumor suppressor PDCD4 (programmed cell death 4), may involve in proliferation and apoptosis pathways. We further verified the potential target PDCD4 and its relevant pathway by luciferase reporter assay combined mutagenesis assay, finding that PDCD4 indeed down-regulated directly by miR-21 overexpression as well as H.pylori infection in AGS cells. According to MTT assay, cell proliferation rate is decreased by PDCD4 overexpression which correlates to its tumor suppressor role. Therefore, we concluded that PDCD4 is down-regulated by miR-21 overexpression and may lead to increased cell proliferation triggered by H.pylori infection in AGS cells.

並列關鍵字

Gastric cancer ； Helicobacter pylori ； miRNAs ； miR-21 ； PDCD4

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