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MicroRNA-200c Increases Radiosensitivity of Human Cancer Cells with Activated EGFR-associated Signaling

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Authors

구태률

Advisor
김인아
Major
의과대학 의학과
Issue Date
2018-02
Publisher
서울대학교 대학원
Keywords
microRNA-200cEGFR-associated signaling networkradiosensitizationhuman cancer cells
Description
학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의학과, 2018. 2. 김인아.
Abstract
Introduction: A member of the microRNA-200 family, microRNA-200c (miR-200c), recently was found to have tumor-suppressive properties by inhibiting the epithelial-mesenchymal transition (EMT) process in several cancers. miR-200c also interacts with various cellular signaling molecules and regulates many important signaling pathways. In the present study, we investigated the radiosensitizing effect of miR-200c and its mechanism of radiosensitization in a panel of human cancer cell lines.
Methods: Malignant glioma (U251, T98G), breast cancer (MDA-MB-468), and lung carcinoma (A549) cell lines were transfected with control pre-microRNA, pre-miR-200c, or anti-miR-200c. Then, RT-PCR, clonogenic assays, immunoblotting, and immunocytochemistry were performed. To predict the potential targets of miR-200c, microRNA databases were used for bioinformatics analysis.
Results: Bioinformatics analysis predicted that miR-200c may be associated with EGFR, AKT2, MAPK1, VEGFA, and HIF1AN. Ectopic overexpression of miR-200c downregulated p-EGFR, p-PI3K, p-AKT, and p-ERK and increased the radiosensitivity of U251, T98G, A549, and MDA-MB-468 cells. In contrast, miR-200c inhibition upregulated p-EGFR, p-PI3K, p-AKT, and p-ERK, and decreased radiation-induced cell killing. miR-200c led to persistent γH2AX focus formation and downregulated pDNA-PKc expression. Autophagy and apoptosis were major modes of cell death. We also confirmed that miR-200c downregulated the expression of VEGF, HIF-1α, and MMP2 in U251 and A549 cells. In these cells, overexpressing miR-200c inhibited invasion, migration, and vascular tube formation. These phenotypic changes were associated with E-cadherin and EphA2 downregulation and N-cadherin upregulation. miR-200c showed no observable cytotoxic effect on normal human fibroblasts and normal human astrocytes.
Conclusions: miR-200c increased the cytotoxic effect of radiotherapy in a panel of human cancers with activated EGFR-associated signaling. The effects of miR-200c on the associated signaling molecules were indirectly confirmed with their phosphorylation levels. miR-200c also mitigated EMT-related processes. Taken together, our data suggest that miR-200c is an attractive target for improving the efficacy of radiotherapy via a unique modulation of the complex regulatory network controlling cancer pro-survival signaling and EMT.
Language
English
URI
https://hdl.handle.net/10371/141028
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