Genome-Wide RNAi Screen to Identify Genetic Interactions with microRNA-26a, and Its Potential Targets

• Main Authors: Yi-Hsuan Chou, 周宜璇
• Other Authors: 林育誼
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/43335235472204846860

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 99 === In post genomic era, a major biological challenge is to observe how genes function as networks to carry out and regulate cellular processes. Genetic interaction analysis is a powerful tool for establishing functional linkages between genes. Hepatocellular carcinoma (HCC) is the third-leading cause of death from cancer and the fifth most common malignancy worldwide. Despite great advances in the diagnosis and treatment of this cancer, relapse and metastasis is largely unavoidable and the 5-year survival rate remains unsatisfactory. MicroRNAs (miRNAs) are a class of highly conserved small RNA molecules that function as critical regulators of gene expression. Mature miRNAs bind to the target mRNAs, resulting in mRNA degradation or translation repression dependent on the sequence complementarily. Importantly, the ability of individual miRNAs to regulate hundreds of mRNAs allows these RNAs to coordinate complex programs of gene expression. Dysregulated miRNA expression has been linked to many human diseases. Previous studies have shown that HCC cells exhibit reduced expression of miR-26a compared to the normal tissue. Activation of nuclear factor κB and interleukin-6 was observed in tumors with reduced miR-26a expression. Moreover, ectopically expressed miR-26a in mouse HCC results in inhibition of cell proliferation and restrains disease progression. These findings indicate that miR-26a may function as a tumor suppressor gene and provide a new approach for HCC treatment. Although the biochemical mechanisms of miRNA have been specified and some candidate miRNA target genes may be predicted by bioinformatics approaches, identification of physiologically relevant targets of individual miRNA remains challenging. In this research, we carried out high-throughput RNA interference screen in human hepatocellular carcinoma cells to identify genes interacting with the query miR-26a gene, which is able to reveal the upstream regulators and downstream effectors. We generated query stable HepG2 cell lines harboring overexpression constructs. We delivered miR26a expression construct into HepG2 cells using lentiviruses followed by TaqMan miRNA assay to confirm the overexpression level. Furthermore, we found that forced expression of miR-26a2 in these stable cell lines render them less capable of growing without anchorage, which is consistent with the proposed tumor suppressor phenotype of this miRNA. Finally, we performed genome-wide genetic interaction screens to identify some interesting genetic interacting partner genes of miR-26a2. These may provide us direct informations on networks, pathways and dynamics of miRNA in hepatocarcinogenesis.