| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 104 === 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.
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