Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level by interacting with the 3'-untranslated region (3'-UTR) of a target gene. To date, over 600 human miRNAs have been identified. Recent miRNA profiling studies reveal that while some miRNAs are overexpressed, the others are underexpressed in tumors compared to normal tissues, suggesting that they could play a key role in oncogenesis. Indeed, functional analyses indicate that miRNAs can act to either promote oncogenesis or suppress tumor suppressors. However, our understanding of miRNA-mediated gene regulation and its impact on oncogenesis is still at an early stage. In this study, we profiled miRNAs in matched breast and colon tumor specimens by real-time RT-PCR and found that mir-145 was underexpressed in both breast and colon tumors compared to the matched normal tissues, suggesting that mir-145 is a potential tumor suppressor gene. To determine the role of mir-145 on tumorogenesis, we cloned the genomic sequence carrying the pre-mir-145 into a CMV promoter driven vector. We found that overexpression of mir-145 was able to inhibit not only tumor cell growth, but also anchorage independent growth in soft agar medium. To further elucidate its role in vivo, we xenografted mice with stably expressing mir-145-HCT116 versus vector control and found that mir-145 can suppress tumor growth significantly. To determine mir-145 target genes, we tested several putative mir-145 targets that are implicated in regulation of cell proliferation and invasion, as suggested by various computer-aided prediction programs. In this regard, we first cloned several UTR's of putative targets into the Luciferase reporter and then verified, through Luciferase assay, that mir-145 can suppress multiple oncogenes involved both in tumor proliferation and metastasis. These targets include MMP-11, MUC-1, ATF-3, ANGPT2, and C-MYC. Although further studies are needed to determine physiological significance of miR-145, these results suggest that mir-145 is a tumor suppressive miRNA which is capable of regulating both tumor growth and invasiveness through suppression of multiple oncogenes.
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