Effect of morpholino-mediated knockdowns of oncofetal RNA-binding proteins on cancer cell biology

Clark, Bethany

Abstract

Background: Muscleblind-like 3 (MBNL3) and insulin-like growth factor 2 binding protein 1 (IGF2BP1) are two RNA-binding proteins that are considered oncofetal genes. The cancer cell-specific expression of oncofetal genes suggests a potential therapeutic target for targeted therapies. Morpholinos are a form of antisense oligonucleotides that have previously been used as a gene target for Duchenne muscular dystrophy.

Aim: This study aimed to design translation-blocking morpholinos to target RNA binding oncofetal genes, IGF2BP1 and MBNL3, and to assess for any anti-cancer effects.

Methods: Human cancer cells (MG-63, SKBR3 and PANC-1) were transfected with morpholinos designed to knock down IGF2BP1 and MBNL3 to determine. Following this, the most effective concentration and time course for achieving significant knockdown was determined. These morpholino doses (1µM and 3µM IGF2BP1.v2 in MG-63 and SKBR3 and 8µM MBNL3.204 in MG-63 and PANC-1) were used for the subsequent experiments. Morpholino knockdown of IGF2BP1 effects on cell biology were measured based on cell survival (Draq7 and Trypan blue staining), cell proliferation (MTT assay), migration (wound healing assay, Boyden chamber) and invasion (in both a cell model and a 3D model). The effect of MBNL3.204 on cancer cell biology were determined with the same assays as IGF2BP1 in addition to the use of spheroid models. MBNL3-regulated alternative splice events were assessed with high-throughput next-generation RNA sequencing, analysed on rMATs and confirmed with PCR.

Results: MG-63 cells transfected with 1µM IGF2BP1.v2 morpholino had a 0.05-fold change in IGF2BP1 expression after 48hrs. This knockdown of IGF2BP1 expression resulted in a significant decrease in MG-63 cell survival and a decrease in cell migration when compared to Wild-type MG-63 and MG-63 cells transfected with a standard control morpholino. The same was seen in SKBR3 cells transfected with 3µM IGF2BP1.v2 morpholino.
MBNL3.204 morpholino resulted in a 0.25- and 0.1-fold change in MBNL3 expression in MG-63 and PANC-1 cells respectively. MBNL3 knockdown resulted in a significant decrease in cancer cell migration and invasion. MBNL3 knockdown resulted in a ~80% decrease in MG-63 spheroid invasion. Changes were also seen in the expression of proteins associated with epithelial-mesenchymal transition (EMT) N-cadherin and E-cadherin consistent with a reduction in EMT when MBNL3 was knocked down. Moreover, MBNL3 knockdown in PANC-1 cells resulted in the expression of alternative splice events occurring in 713 genes, including the expression of pro-apoptotic isoforms of APAF1, CASP8 and BAG6, despite no significant change to cell death and apoptosis in cell biology assays.

Conclusion: The knockdown of IGF2BP1 and MBNL3 with translation-blocking morpholinos offers a potential option for an anti-cancer therapeutic as there was a reduction in critical aspects of cancer progression, cell survival and migration, potentially providing an alternative to current cancer treatments that has less adverse systemic effects, thereby improving the quality of life for patients.

• Thesis Type: Thesis
• Deposit Date: Nov 3, 2023
• Publicly Available Date: Aug 30, 2024
• Public URL: https://uwe-repository.worktribe.com/output/11407736
• Award Date: Aug 30, 2024