Effect of short activating RNA AW1-51 on IRES-dependent CEBPA translation
Embargo Date
2027-09-22
OA Version
Citation
Abstract
DNA methylation is an important epigenetic modification of DNA involved in the regulation of gene expression by affecting the chromatin structure. DNA methyltransferases (DNMTs) are involved in the methylation of CpG-rich islands which leads to repression of the affected gene. Methylation of tumor suppressor genes is the most prevalent cause of malignant cancers. CEBPA is a gene that encodes the transcription factor CEBPA, which is involved in myeloid cell differentiation and plays a tumor-suppressive role. Short-activating RNAs (saRNA) can be used to treat certain cancers by inducing gene expression. Our lab had previously observed that the saRNA, AW1-51, is capable of inducing CEBPA expression in K562 and similarly in A549 cell lines by influencing the DNA methylation levels of its promoter region. Remarkably, we detected a considerable increase of CEBPA protein expression by AW1-51 that preceded the transcriptional activation in A549, wherein CEBPA protein can be induced in contrast to K562.
We therefore hypothesized a potential role for AW1-51 as a translational activator, in addition to the most well-reported transcriptional effect. Consistently, analyses carried out to verify this hypothesis did point to a mechanism whereby AW1-51 would promote a non-cap-mediated translation of the CEBPA protein, implying a novel mode of action for this RNA previously unknown.
We decided to generate a reporter system that included a GFP fluorescent marker to assess non-cap mediated translation, an mCherry fluorescent protein to follow cap-mediated translation, and various inserts to test our hypothesis. We transfected the reporter system into A549 cells then transfected the cells with AW1-51 and Fluc (negative control) and analyzed the results by microscope imaging and flow cytometry. Our results did not lead to conclusive results and further validations of the reporter system’s efficiency will be needed to delve into this matter.
Description
2024