Hypoxia mediates cancer development and progression through HIF-1a and microRNA regulation

Background: MicroRNAs (miRNAs) are small non coding RNAs which play a role in several cellular processes such as apoptosis, cell differentiation, proliferation and stress response. Thus, changes in miRNA expression are associated with the development and progression of human disease like cancer and many other systemic diseases. MiRNAs expression could be influenced by different stimuli such as oxidative stress, inflammatory response and hypoxia. Lately, some hypoxia-inducible miRNAs (HRMs, hypoxia-regulated miRNAs) have been identified. These HRMs are often activated in different types of cancers, suggesting their role in tumorigenesis. Obstructive sleep apnea (OSA) is a breathing disorder characterized by recurrent obstructions of the upper airway associated with increased inspiratory efforts, intermittent hypoxemia and sleep fragmentation. Recent experiments suggest that cancer could be a disease associated with this sleep breathing disorder. The mechanisms that regulate gene expression during hypoxia are not fully understood but miRNAs expression seems to have an important role in various processes. On these basis, the main aims of this experimental thesis are: i) to evaluate the roles of continuous hypoxia (CH) and miRNAs in cancer; ii) to evaluate how hypoxia could influence the expression of specific miRNAs in cells; iii) to assess the differences in the expression profile of specific miRNAs induced by continuous and intermittent hypoxia; iv) to evaluate changes in miRNAs expression in controls or patients affected by OSA and/or cancer and in colorectal cancer cells exposed to intermittent hypoxia (IH), and to evaluate their impact on tumor progression in vitro. Materials and Methods: For this purpose, I used qRT-PCR to detect miRNAs expression both CaCo2 cells exposed to CH or IH, to 2 to 24/32h, with or without acriflavine (ACF), a HIF-1 inhibitor and in patients’ sera. MTT and transwell invasion test were applied to investigate the proliferation and migration of CaCo2 exposed to IH and treated with miRNA inhibitors or ACF. HIF-1 activity was evaluated in CaCo2 cells after IH. Results: The levels of almost all analyzed miRNAs (miR-21, miR-23b, miR-26a, miR-27b, and miR- 145) were greater in continuous hypoxia versus normoxia. Furthermore, the expression of nearly all miRNAs was higher in IH than in CH. In addition, the levels of miR-21, miR-26a and miR-210 increased in OSA and ONCO-OSA patients compared to controls. MiR-23b increased in ONCO-OSA patients, and miR-27b and miR-145 increased in OSA but not ONCO-OSA patients. MiR-21, miR-26a, miR-23b and miR-210 increased in cells after IH. Proliferation and migration were increased by IH and reduced after either miRNA inhibition or ACF treatment. MiRNA inhibition reduces HIF-1α gene expression. Conversely, the expression of these miRNAs was significantly decreased after ACF treatment. Conclusions: We identified a series of miRNAs, induced by the hypoxic environment, in malignancies associated with hypoxia, especially OSA-associated intermittent hypoxia. These data would demonstrate that there is a different response between the condition of intermittent versus chronic hypoxia, and the former appears to be much more harmful in terms of stimulation of the expression of some miRNAs. These miRNAs could be implicated in cancer development and progression through a regulatory loop involving HIF-1.