Functional analysis of non-viral miRNA replacement therapy in colon carcinoma in vitro and in vivo
MicroRNAs (miRNAs) are small RNA molecules with perfect or imperfect homology to their target mRNA. They have been shown to specifically interfere with the expression of their target protein(s). Moreover, microRNAs play an important role in the pathogenesis of cancer, where some microRNAs act as...
Pharmakologie und Toxikologie
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|Zusammenfassung:||MicroRNAs (miRNAs) are small RNA molecules with perfect or imperfect homology to their target mRNA. They have been shown to specifically interfere with the expression of their target protein(s). Moreover, microRNAs play an important role in the pathogenesis of cancer, where some microRNAs act as tumor suppressors by targeting lethal oncogenes, and others act as oncogenes by targeting suppressor genes. Thus, microRNAs can be involved directly or indirectly in important cellular processes such as proliferation, differentiation and apoptosis. Understanding the functions and importance of microRNAs could lead to the discovery of new strategies for controlling of cancer. This thesis was aiming at the possibility of using non-viral delivery systems for microRNAs into mammalian cell lines, and examined their efficacies. To this end, three different transfection reagents, linear Jet-PEI, branched PEI F25-LMW and INTERFERin, were used in vitro, either commercially available or prepared in our lab. The work was concentrating on colon cancer as a tumor model, and two representative cell lines, LS174t and HCT-116, were selected as a platform to conduct the experiments. The studies focused on miR-143 and miR-145, which had been implicated previously with cancer development and progression. It was found in miRNA transfection experiments that miR-143 and miR-145, which are downregulated in colon cancer, have the ability to inhibit the progression of the cancer through different regulatory mechanisms, proliferation and apoptosis. Some important genes, which play important roles in tumorigenesis and tumor progression, were found to be negatively regulated by both miR-143 and miR-145. More specifically, ERK5 (Mitogen Activated Protein Kinase) which plays an essential role in the proliferation of colon cancer was suppressed by miR-143 and miR-145. Likewise, c-Myc was negatively regulated by miR-145, but these results were only at the protein level, i.e., no significant effects were determined on c-Myc transcription. Furthermore, it was found that the^inhibitory effects were depending on several factors such as cell line, transfection reagent, time of incubation and concentration of miRNA complex. Thus, the cellular delivery of microRNAs miR-143 and miR-145 showed profound inhibitory effects on the proliferation and soft agar colony formation of LS174t and HCT- 116 cells, and induced apoptosis in LS174t cells. Moreover, miR-145 was examined for its tumor inhibitory effect in vivo. To this end, athymic nude mice bearing s.c. colon carcinoma xenografts were treated with PEIcomplexed miR-145 by systemic or local injection. Profound anti-tumor effects upon PEImediated miR-145 delivery were found which were again dependent on the downregulation of specific target genes. Taken together, microRNA replacement therapy may represent a promising approach in tumor treatment, and this thesis establishes the PEI-mediated therapeutic delivery of miR- 145 in colon carcinoma.|