Poster abstracts
Poster number 106 submitted by Chloe Nagasawa
Micromanaging the Message: Distinct roles of miR-29 family members in MDM2 splicing and tumor stress response
Chloe K. Nagasawa (Molecular Medicine and Therapeutics, The Ohio State University), Matias Montes (Stanford University), Dawn S. Chandler (Molecular Medicine and Therapeutics, The Ohio State University)
Abstract:
Mouse double minute 2 homolog (MDM2) is an oncogene and critical negative regulator of tumor suppressor p53. In rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, ~75% of tumors express MDM2-ALT1, an alternatively spliced isoform linked to high-risk disease and tumorigenesis. MDM2-ALT1 arises from the skipping of eight exons (4–11) and ligation of exons 3 and 12, producing a truncated protein that disrupts p53 regulation. Despite its clinical significance, the splicing mechanisms remain poorly understood.
We recently identified miR-29b as a novel regulator of MDM2 splicing. MiR-29b levels are reduced in RMS tumors and cell lines, suggesting a role in pathogenesis. Here, we explore the broader role of the miR-29 family – miR-29a, miR-29b and miR-29c – in MDM2 regulation. All share a seed sequence, but only miR-29b has a nuclear localization sequence. While all are predicted to bind the MDM2 3’UTR, only miR-29a and miR-29b are found in the supraspliceosome. Given these findings, we aimed to determine how miR-29a and miR-29b differentially regulate MDM2 expression and splicing.
Using an inducible genotoxic stress system (UV-C/cisplatin) in Rh30 cells, we observed decreased full-length MDM2 and increased MDM2-ALT1, mirroring the splicing in RMS tumors and cell lines. With this system, we observed that miR-29b expression decreased, while miR-29a levels remain unchanged. To explore this further, we overexpressed miR-29a or miR-29b in Rh30 cells treated with or without cisplatin. While both miR-29a and miR-29b significantly reduced total MDM2 expression compared to a control, only miR-29b decreased MDM2-ALT1, shifting the ratio of full-length to MDM2-ALT1. Moreover, our luciferase assays revealed that miR-29a and miR-29b regulate MDM2 via canonical miRNA targeting. These findings demonstrate that while miR-29a and miR-29b downregulate MDM2, miR-29b uniquely modulates its splicing under stress, highlighting its potential as a therapeutic target in RMS.
Keywords: splicing, microRNA