| Summary: | 碩士 === 國立陽明大學 === 藥理學研究所 === 103 === In the past two decades, numerous studies have identified various somatic mitochondrial DNA (mtDNA) mutations in 62.4% of examined primary human breast cancers. Patients with mtDNA mutations were associated with an older onset age, a higher histological stage and poor prognosis. Nevertheless, the causal role for mtDNA mutations in breast cancer is still unclear. The aim of this study was to explore the effect of mtDNA mutations on human breast cancer by allotopic expression system. In previous study, the constructs of different length fragments of the nuclear gene SIRT3 (as mitochondrial targeting sequence, MTS) fused with the recoded mutant mitochondrial gene shortND5 were successfully expressed in HEK293T cells. In this study, the shortND5 was replaced with the recoded longND5 or mutant MTATP6 but they were not expressed except 300SIRT-longND5. The plasmids containing both MTS and the 3’UTR of SOD2 failed to enhance protein expression compared with the MTS alone. However, this study confirmed that the shortND5 and longND5 successfully produced in HEK293T cells could be translocated into mitochondria. There were no significant differences between the mutant mitochondrial genes and empty vector in ROS production, ATP content, lactate production and oxygen consumption rate. But the cells transfected with shortND5 or longND5 gained a decreased migratory phenotype. Due to the used poor transfection efficiency for breast cancer cells in this study, it was difficult to detect the protein expressions of the recombinant mutant genes in breast cancer cells. Under a condition with higher transfection efficiency, the recombinant proteins were detectable in human breast cancer HCC1937 cells. These findings demonstrated mutant ND5 gene might repress HEK293T cells migration. The effects of the mitochondrial mutant genes on HCC1937 breast cancer cells need to be further investigated. The method for expressing mitochondrial genes using the allotopic expression system still needs to be further improved.
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