| Summary |
Cancer is a prevalent disease that affects millions of people each year across the globe. In an effort to find therapies to the many types of cancers, regulation of protein expression through transcription and translation pathways have been extensively studied. However, one area which has been often overlooked is the importance of post-transcriptional regulation in protein output. The goal of this dissertation project has been to understand post-transcriptional regulation of mRNA during a cellular stress that all cancers must overcome in order to survive, oxygen deprivation. The lack of oxygen, termed hypoxia, is known to affect tumor growth and angiogenesis. Specifically, hypoxia affects the post-transcriptional regulation of mRNAs by increasing the stability of a subset of ischemia-related mRNAs, including VEGF. Multiple factors including RNA binding proteins and miRNAs have been identified to be important for the post-transcriptional regulation of individual mRNAs, but mechanisms regulating global stability have not been elucidated. Recently, the mRNA modification, N⁶ methyladenosine (m6A), has been shown to be involved in the post-transcriptional regulation processes of mRNA stability and promotion of translation. Therefore, I set out to investigate the effect of hypoxia on RNA m6A content. My results show that hypoxic exposure leads to striking changes in the m6A content of mRNA in HEK-293T cells as well as immortalized and oncogenically transformed human mammary epithelial cells (HMECs). Using m6A mRNA immunoprecipitation, we identified a number of specific hypoxia related mRNAs, including Glut1 and c-Myc, which show increased m6A levels under hypoxic conditions. Many of these same mRNAs also exhibit increased mRNA stability revealed by metabolic labeling of RNA using 4sU. Furthermore, knockdown of the m6A-specific methyltransferases METTL3/14 blocked the hypoxic stabilization of these mRNA. The increase in mRNA stability through m6A led to greater translational efficiency after recovery from the hypoxic stress. Overexpressing m6A in oncogenically transformed HMEC in normal oxygen conditions led to an increase in would healing, proliferation, and invasion abilities. Ultimately, the mRNA modification, m6A, led to phenotypic changes in a cancer cell, and it may be possible to manipulate this mRNA modification in order to slow cancer growth. |
