Abstract 14635: Hypoxia Regulates the Trafficking of microRNAs in Endothelial Cells
MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression by translational repression or mRNA degradation. They are associated with diverse functions in physiological processes and diseases.
Whereas mRNAs are known to traffic in cells to mediate local protein synthesis, it is unclear whether the intracellular localization of miRs may be regulated under stress conditions. Therefore, we investigated the intracellular localization and trafficking of miRs under basal conditions and upon exposure to hypoxia (0.2 % O2) for 24 hours.
We isolated cellular compartments by sucrose gradient centrifugation and detected the expression of the endothelial enriched miR-126 and miR-92a, the anti-atherosclerotic miR-145 and the matrix regulating miR-29b in the individual fractions by real-time PCR. Western blots with marker proteins for typical cell compartments indicated highest concentrations of miRs in the fractions that show markers for endoplasmatic reticulum (ER) and golgi apparatus. Interestingly, application of hypoxia resulted in a significant decrease of miR-92a (73±11%), miR-126 (80±11%) and miR-145 (70±12%) in the ER/golgi fraction, whereas the overall expression of the miRs was not reduced. In contrast to all other miRs tested, miR-29b was highly detected in the nuclear fraction and significantly reduced in this fraction by 70±8% after hypoxia exposure.
To determine the trafficking of miRs by life imaging, we further developed molecular beacons that emit fluorescence only after specifically binding to miR-92a. We confirmed the results obtained by sucrose gradients and showed that the localization of miR-92a to the golgi apparatus was reduced by hypoxia (67±8%). To determine whether the reduced transport of miRs to the ER/golgi apparatus regulates the miR export we measured the miRs in the cell supernatants. Preliminary data indicate a reduced export of miRs by hypoxia.
In summary, these data suggest that hypoxia reduces the intracellular trafficking of miRs to the ER/golgi apparatus resulting in a reduced export of miRs. The modulation of miR trafficking might have implications for the regulation of miR targets and intercellular communication.
- © 2013 by American Heart Association, Inc.